Crystalline forms of therapeutic compounds and uses thereof

ABSTRACT

Described herein are certain crystalline forms of Compound 3, as well as pharmaceutical compositions employing the crystalline forms. Also provided are particles (e.g., nanoparticles) comprising such crystalline forms or pharmaceutical compositions. In certain examples, the particles are mucus penetrating particles (MPPs). The present invention further relates to methods of treating or preventing diseases using crystalline forms or pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/806,819, filed Mar. 2, 2020, now U.S. Pat. No. 10,975,090, which is acontinuation of U.S. patent application Ser. No. 16/184,760 filed Nov.8, 2018, now U.S. Pat. No. 10,618,960, which is a continuation of U.S.patent application Ser. No. 15/712,645 filed Sep. 22, 2107, now U.S.Pat. No. 10,160,765, which is a continuation of U.S. patent applicationSer. No. 14/981,105, filed Dec. 28, 2015, now U.S. Pat. No. 9,790,232,which is a division of U.S. patent application Ser. No. 14/530,092 filedOct. 31, 2014, now U.S. Pat. No. 9,458,169, which claims the benefit ofU.S. Provisional Patent applications 61/898,741 filed Nov. 1, 2013,62/039,177 filed Aug. 19, 2014, and 62/039,192 filed Aug. 19, 2014. Theentire contents of all of these applications are incorporated byreference herein.

FIELD OF THE INVENTION

This invention relates to crystalline forms of a therapeutic compounduseful for treating diseases, including proliferative diseases anddiseases associated with angiogenesis, such as cancer and maculardegeneration.

BACKGROUND OF THE INVENTION

Growth factors play an important role in angiogenesis,lymphangiogenesis, and vasculogenesis. Growth factors regulateangiogenesis in a variety of processes including embryonic development,wound healing, and several aspects of female reproductive function.Undesirable or pathological angiogenesis is associated with diseasesincluding diabetic retinopathy, psoriasis, cancer, rheumatoid arthritis,atheroma, Kaposi's sarcoma, and hemangioma (Fan et al., 1995, TrendsPharmacol. Sci. 16: 57 66; Folkman, 1995, Nature Medicine 1: 27 31).Angiogenic ocular conditions represent the leading cause of irreversiblevision loss in developed countries. In the United States, for example,retinopathy of prematurity, diabetic retinopathy, and age-relatedmacular degeneration are the principal causes of blindness in infants,working age adults, and the elderly, respectively. Efforts have beendeveloped to inhibit angiogenesis in the treatment of these conditions(R. Roskoski Jr., Critical Reviews in Oncology/Hematology, 62 (2007),179-213).

Therefore, there is a need for new therapeutic compounds for thetreatment of diseases associated with the aberrant signaling of growthfactors and diseases associated with angiogenesis, such as cancer,macular degeneration, and diabetic retinopathy.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to crystalline forms ofcompound7-(3-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinazolin-7-yloxy)propyl)-2-oxa-7-azaspiro[3.5]nonane,referred to herein as Compound 3 and shown below:

In one embodiment, the present invention is Compound 3 depicted above,7-(3-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinazolin-7-yloxy)propyl)-2-oxa-7-azaspiro[3.5]nonane, in crystalline Form A. In certain embodiments, the crystallineform is crystalline Form A having an X-Ray Powder Diffraction (XRPD)pattern with peaks at about 6.11, 9.63, 16.41, 18.60, 20.36 and23.01±0.3 degrees two theta or 14.45, 9.17, 5.40, 4.77, 4.36 and3.86±0.3 Å in d-spacing. In further embodiments, crystalline Form Afurther has XRPD peaks at about 11.46, 12.26, 18.16, 19.51, 21.12 and25.71±0.3 degrees two theta or 7.71, 7.22, 4.88, 4.55, 4.20 and 3.46±0.3Å in d-spacing. In further embodiments, crystalline Form A further hasXRPD peaks at about 11.10, 15.66, 17.54, 22.31, 24.79 and 28.90±0.3degrees two theta or 7.96, 5.65, 5.05, 3.98, 3.59 and 3.09±0.3 Å ind-spacing. In still further embodiments, crystalline Form A has an XRPDpattern with peaks at about 6.11, 9.63, 11.10, 11.46, 12.26, 15.66,16.41, 17.54, 18.16, 18.60, 19.51, 20.36, 21.12, 22.31, 23.01, 24.79,25.71 and 28.90±0.3 degrees two theta or 14.45, 9.17, 7.96, 7.71, 7.22,5.65, 5.40, 5.05, 4.88, 4.77, 4.55, 4.36, 4.20, 3.98, 3.86, 3.59, 3.46and 3.09±0.3 Å in d-spacing.

In other embodiments, the present invention provides7-(3-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinazolin-7-yloxy)propyl)-2-oxa-7-azaspiro[3.5]nonane,in crystalline Form B. In certain embodiments, the crystalline form iscrystalline Form B having an X-Ray Powder Diffraction (XRPD) patternwith peaks at about 7.70, 13.53, 17.27, 18.44, 19.73, 23.10 and26.07±0.3 degrees two theta or 11.47, 6.54, 5.13, 4.81, 4.50, 3.85 and3.41±0.3 Å in d-spacing. In further embodiments, crystalline Form Bfurther has XRPD peaks at about 9.87, 12.88, 14.40, 15.45, 21.14 and26.84±0.3 degrees two theta or 8.96, 6.87, 6.14, 5.73, 4.20 and 3.32±0.3Å in d-spacing. In further embodiments, crystalline Form B further hasXRPD peaks at about 10.69, 16.42, 18.90, 22.56, and 29.12±0.3 degreestwo theta or 8.27, 5.39, 4.69, 3.94 and 3.06±0.3 Å in d-spacing. Instill further embodiments, crystalline Form B has an XRPD pattern withpeaks at about 7.70, 9.87, 10.69, 12.88, 13.53, 14.40, 15.45, 16.42,17.27, 18.44, 18.90, 19.73, 21.14, 22.56, 23.10, 26.07, 26.84 and29.12±0.3 degrees two theta or 11.47, 8.96, 8.27, 6.87, 6.54, 6.14,5.73, 5.39, 5.13, 4.81, 4.69, 4.50, 4.20, 3.94, 3.85, 3.41, 3.32 and3.06±0.3 Å in d-spacing.

In one aspect, the present invention relates to a compound having theformula

in crystalline Form A.

In another aspect, the present invention relates to a crystalline formof a compound having the formula

wherein said crystalline form is crystalline Form A having an X-raypowder diffraction (XRPD) pattern with peaks at about 6.11, 9.63, 16.41,18.60, 20.36 and 23.01±0.3 degrees two theta, or 14.45, 9.17, 5.40,4.77, 4.36 and 3.86±0.3 Å in d-spacing.

In another embodiment, the present invention relates to a compoundhaving the formula

in crystalline Form B.

In another embodiment, the present invention relates to a crystallineform of a compound having the formula

wherein said crystalline form is crystalline Form B having an X-RayPowder Diffraction (XRPD) pattern with peaks at about 7.70, 13.53,17.27, 18.44, 19.73, 23.10 and 26.07±0.3 degrees two theta or 11.47,6.54, 5.13, 4.81, 4.50, 3.85 and 3.41±0.3 Å in d-spacing.

In another aspect, the present invention relates to a process forpreparing a crystalline form of Compound 3. In certain embodiments, thepresent invention relates to a method for preparing crystalline Form Aof Compound 3. In additional embodiments, the method of preparingcrystalline Form A comprises wet-milling a slurry comprising anamorphous form of Compound 3 and a non-ionic surfactant to obtainnanoparticles of the compound. In further embodiments, the resultingnanoparticles of crystalline Form A have an XRPD pattern with peaks atabout 6.11, 9.63, 16.41, 18.60, 20.36 and 23.01±0.3 degrees two theta or14.45, 9.17, 5.40, 4.77, 4.36 and 3.86±0.3 Å in d-spacing. In furtherembodiments, crystalline Form A further has XRPD peaks at about 11.46,12.26, 18.16, 19.51, 21.12 and 25.71±0.3 degrees two theta or 7.71,7.22, 4.88, 4.55, 4.20 and 3.46±0.3 Å in d-spacing. In furtherembodiments, crystalline Form A further has XRPD peaks at about 11.10,15.66, 17.54, 22.31, 24.79 and 28.90±0.3 degrees two theta or 7.96,5.65, 5.05, 3.98, 3.59 and 3.09±0.3 Å in d-spacing. In still furtherembodiments, crystalline Form A has an XRPD pattern with peaks at about6.11, 9.63, 11.10, 11.46, 12.26, 15.66, 16.41, 17.54, 18.16, 18.60,19.51, 20.36, 21.12, 22.31, 23.01, 24.79, 25.71 and 28.90±0.3 degreestwo theta or 14.45, 9.17, 7.96, 7.71, 7.22, 5.65, 5.40, 5.05, 4.88,4.77, 4.55, 4.36, 4.20, 3.98, 3.86, 3.59, 3.46 and 3.09±0.3 Å ind-spacing.

In other embodiments, the present invention relates to a method forpreparing crystalline Form B of Compound 3. In certain embodiments, themethod of preparing crystalline Form B comprises a) dissolving theamorphous form of Compound 3 in water and acetone; b) crystallizingCompound 3 from a solvent mixture comprising water and acetone; and c)isolating the crystalline Form B of Compound 3 from the solvent mixture.In certain embodiments, the starting Compound 3 is amorphous. Inparticular embodiments, the method of preparing crystalline Form Butilizes a solvent mixture consisting of 4:1 acetone:water. In otherembodiments, the method of preparing crystalline Form B furthercomprises the step of heating the solvent mixture to dissolve thecompound and/or cooling the solvent mixture to allow crystal formation.In some embodiments, the resulting crystalline Form B has an XRPDpattern with peaks at about 7.70, 13.53, 17.27, 18.44, 19.73, 23.10 and26.07±0.3 degrees two theta or 11.47, 6.54, 5.13, 4.81, 4.50, 3.85 and3.41±0.3 Å in d-spacing. In further embodiments, crystalline Form Bfurther has XRPD peaks at about 9.87, 12.88, 14.40, 15.45, 21.14 and26.84±0.3 degrees two theta or 8.96, 6.87, 6.14, 5.73, 4.20 and 3.32±0.3Å in d-spacing. In further embodiments, crystalline Form B further hasXRPD peaks at about 10.69, 16.42, 18.90, 22.56, and 29.12±0.3 degreestwo theta or 8.27, 5.39, 4.69, 3.94 and 3.06±0.3 Å in d-spacing. Instill further embodiments, crystalline Form B has an XRPD pattern withpeaks at about 7.70, 9.87, 10.69, 12.88, 13.53, 14.40, 15.45, 16.42,17.27, 18.44, 18.90, 19.73, 21.14, 22.56, 23.10, 26.07, 26.84 and29.12±0.3 degrees two theta or 11.47, 8.96, 8.27, 6.87, 6.54, 6.14,5.73, 5.39, 5.13, 4.81, 4.69, 4.50, 4.20, 3.94, 3.85, 3.41, 3.32 and3.06±0.3 Å in d-spacing.

In yet another aspect, the present invention relates to pharmaceuticalcompositions and kits to treat diseases, including proliferativediseases, ocular diseases, dermatological diseases, inflammatorydiseases, autoimmune diseases, auto-inflammatory diseases, and metabolicdiseases comprising a crystalline form of Compound 3. In a furtheraspect, the present invention provides methods of using a crystallineform of Compound 3 to study the inhibition of growth factor signalingand/or to treat and/or prevent proliferative diseases, ocular diseases,dermatological diseases, inflammatory diseases, autoimmune diseases,auto-inflammatory diseases, and metabolic diseases. In certainparticular aspects, a crystalline form of Compound 3 is used in treatingdiseases associated with angiogenesis.

In another aspect, the present invention provides pharmaceuticalcompositions comprising crystalline forms of Compound 3, wherein thepharmaceutical compositions optionally comprise a pharmaceuticallyacceptable carrier. In certain embodiments, the pharmaceuticalcompositions described herein include a therapeutically effective amountof a crystalline form of Compound 3. In certain embodiments, thepharmaceutical composition may be useful for treating proliferativediseases (e.g., cancers, benign neoplasms, inflammatory diseases,autoimmune diseases) and/or ocular diseases (e.g., macular degeneration,glaucoma, diabetic retinopathy, retinoblastoma, edema, uveitis, dry eye,blepharitis, and post-surgical inflammation) in a subject in needthereof. The pharmaceutical composition may also be useful forinhibiting abnormal angiogenesis and/or aberrant signaling of a growthfactor in a subject or cell.

In some embodiments, the crystalline forms of Compound 3 may be intendedfor delivery in a subject's tissues having mucus (e.g., eye, respiratorytract, gastrointestinal tract, genito-urinary tract), which is aviscoelastic and adhesive substance that traps most foreign objects(e.g., microorganisms, particles, dust). Compound or particles that areimmobilized in the mucus are quickly eliminated by mucus clearancemechanisms; therefore, they are not able to effectively deliver theintended therapeutic effect. In these tissues, for the compound toeffective, it must quickly penetrate the mucus and/or avoid mucusclearance mechanisms. Accordingly, modifying mucoadhesive compounds orparticles containing compounds with a coating to reduce themucoadhesiveness, and decreasing the size of the particles of compoundmay allow for efficient delivery and therapeutic effect.

In one aspect of the invention, the crystalline forms of Compound 3 ofthe invention are formulated into mucus penetrating particles or mucuspenetrating crystals (collectively, MPPs) suitable for administration(e.g., topical, inhalation, injection) to tissues of the subject havingmucus (e.g., eye, respiratory tract, gastrointestinal tract,genito-urinary tract). In certain embodiments, the particles comprisinga crystalline form of Compound 3 (e.g., crystalline Form B) are mucuspenetrating. The MPPs may include a coating surrounding a core. The coremay contain primarily a crystalline form of Compound 3, or the core maybe a polymeric core with the crystalline form of Compound 3 encapsulatedin the polymer. In certain embodiments, the MPPs are nanoparticles(e.g., particles having an average diameter of at least about 10 nm andless than about 1 μm). The MPPs may be useful in delivering thepharmaceutical agent to a subject. In certain embodiments, the MPPs arecapable of delivering the crystalline form of Compound 3 in or throughmucus of a subject.

Another aspect of the invention relates to pharmaceutical compositionscomprising particles comprising crystalline forms of Compound 3. In oneparticular embodiment, the particles comprise crystalline Form B ofCompound 3. In another embodiment, the particles comprise crystallineForm A of Compound 3. In certain embodiments, the pharmaceuticalcompositions are useful in delivering crystalline forms of Compound 3 toa subject.

In another aspect of the invention, the present invention providespharmaceutical compositions comprising a plurality of particlescomprising (i) a core comprising a crystalline form of Compound 3, and(ii) a coating of a surface altering agent surrounding the core, whereinthe surface altering agent is present on the outer surface of the coreat a density of at least 0.01 surface altering agent per nm², andoptionally, at least one pharmaceutically acceptable excipient. In someembodiments, the surface altering agent is a triblock copolymer of thestructure (hydrophilic block)-(hydrophobic block)-(hydrophilic block).In some aspects, the triblock copolymer is a PLURONIC® or poloxamer. Inother aspects, the surface altering agent is a poly(vinyl alcohol) or apolysorbate. In one preferred aspect, the core comprises crystallineForm B of Compound 3. In another, the core comprises crystalline Form Aof Compound 3

In certain embodiments, the compound, particle, or pharmaceuticalcomposition is formulated to be mucus penetrating.

Another aspect of the present invention relates to methods of treatingand/or preventing a disease associated with abnormal angiogenesis in asubject in need thereof.

Another aspect of the present invention relates to methods of treatingand/or preventing a disease associated with aberrant signaling of agrowth factor signaling pathway in a subject in need thereof.

In another aspect, the present invention provides methods of inhibitingangiogenesis in a subject in need thereof.

In another aspect, the present invention provides methods of inhibitingaberrant signaling of a growth factor signaling pathway in a subject orcell. In certain embodiments, the growth factor is associated withangiogenesis. In certain embodiments, the growth factor is VEGF.

The methods of the present invention include administering to a subjectan effective amount of a crystalline form of Compound 3 orpharmaceutical compositions thereof of the invention. The diseasesinclude proliferative diseases, ocular diseases, dermatologicaldiseases, inflammatory diseases, autoimmune diseases, autoinflammatorydiseases, and metabolic diseases. In certain embodiments, the effectiveamount is a prophylactically effective amount.

In another aspect, the present invention provides kits comprising acrystalline form of Compound 3. The kits of the invention may include asingle dose or multiple doses of a crystalline form of Compound 3, orpharmaceutical compositions thereof. The provided kits may be useful forthe treatment of proliferative diseases, ocular diseases, dermatologicaldiseases, inflammatory diseases, autoimmune diseases, autoinflammatorydiseases, and metabolic diseases. In certain embodiments, the kitsdescribed herein may be useful in treating and/or preventing a diseaseassociated with abnormal angiogenesis and/or with aberrant signaling ofa growth factor in a subject in need thereof. The kits may also beuseful for inhibiting abnormal angiogenesis and/or aberrant signaling ofa growth factor signaling pathway in a subject in need thereof. Incertain embodiments, the kit further includes instructions foradministering crystalline forms of Compound 3 of the invention. The kitsmay also include packaging information describing the use or prescribinginformation for the subject or a health care professional. Suchinformation may be required by a regulatory agency such as the U.S. Foodand Drug Administration (FDA). The kit may also optionally include adevice for administration of Compound 3 crystalline forms or compositionthereof, for example, a dropper for ocular administration or a syringefor parenteral administration.

The details of one or more embodiments of the invention are set forthherein. Other features, objects, and advantages of the invention will beapparent from the Detailed Description, the Figures, the Examples, andthe Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a representative X-Ray Powder Diffraction (XRPD) patternfor crystalline Form A of Compound 3.

FIG. 2 provides a representative XRPD pattern for crystalline Form B ofCompound 3.

FIG. 3 provides a representative Differential Scanning Calorimetry (DSC)thermogram for crystalline Form B of Compound 3.

FIG. 4 provides a representative Thermogravimetric Analysis (TGA)thermogram for crystalline Form B of Compound 3.

FIG. 5 provides an XRPD pattern for crystalline Form B of Compound 3after its formation (bottom trace) and after being held in suspensionfor 7 weeks (top trace).

FIG. 6 provides an XRPD pattern for crystalline Form B of Compound 3(bottom trace) and an XRPD pattern of a milling mixture of amorphousCompound 3 and crystalline Form B of Compound 3 (top trace).

FIG. 7 provides XRPD patterns, from bottom to top, for: crystalline FormA of Compound 3, crystalline Form B of Compound 3, a mixture ofcrystalline Forms A and B that were milled separately and then combined(t=0), a mixture of crystalline Forms A and B that were milledseparately and then combined and stored at room temperature for >2months, and a mixture of crystalline Forms A and B that were milledseparately and then combined and stirred at room temperature for 5weeks.

FIG. 8 is a PK profile for Compound 3 in choroid tissue of Gottingenmini-pig after topical administration.

FIG. 9 is a PK profile for Compound 3 in retina tissue of Gottingenmini-pig after topical administration.

FIG. 10 is a PK profile for Compound 3 in plasma of Gottingen mini-pigafter topical administration.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

As used herein, when referring to X-Ray Powder Diffraction (XRPD) peakpositions, “about” means ±0.3, preferably ±0.2, more preferably ±0.1,more preferably ±0.05, and still more preferably ±0.02

OTHER DEFINITIONS

The following definitions are more general terms used throughout thepresent application.

The term “polymorphs” refers to a crystalline form of a compound (or asalt, hydrate, or solvate thereof) in a particular crystal packingarrangement. All polymorphs have the same elemental composition.Different crystalline forms usually have different X-ray diffractionpatterns (e.g., XRPD patterns), infrared spectra, melting points,density, hardness, crystal shape, optical and electrical properties,stability, and/or solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. One particular method for characterizingdifferent crystalline forms of a compound is X-Ray Powder Diffraction(XRPD) analysis, which is a technique that is well-known in the art.Various polymorphs of a compound can be prepared by crystallizationunder different conditions. As used herein, the term “crystal form” or“crystalline form” refers to one particular polymorph of a compound thatpossesses one or more particular identifying characteristics, forexample, a particular X-ray diffraction or XRPD pattern.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g., infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult, or senior adult)) and/or othernon-human animals, for example, mammals (e.g., primates (e.g.,cynomolgus monkeys, rhesus monkeys); commercially relevant mammals suchas cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds(e.g., commercially relevant birds such as chickens, ducks, geese,and/or turkeys). In certain embodiments, the animal is a mammal. Theanimal may be a male or female at any stage of development. The animalmay be a transgenic animal or genetically engineered animal. In certainembodiments, the subject is a non-human animal. In certain embodiments,the animal is fish. A “patient” refers to a human subject in need oftreatment of a disease.

The terms “administer,” “administering,” or “administration,” as usedherein, refer to implanting, absorbing, ingesting, injecting, inhaling,or otherwise introducing a crystalline form of Compound 3, or apharmaceutical composition thereof, in or on a subject.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease described herein. In some embodiments, treatmentmay be administered after one or more signs or symptoms of the diseasehave developed or have been observed. In other embodiments, treatmentmay be administered in the absence of signs or symptoms of the disease.For example, treatment may be administered to a susceptible subjectprior to the onset of symptoms (e.g., in light of a history of symptomsand/or in light of genetic or other susceptibility factors) or exposureto a pathogen). Treatment may also be continued after symptoms haveresolved, for example, to delay or prevent recurrence.

As used herein, the terms “condition,” “disease,” and “disorder” areused interchangeably.

An “effective amount” of a Compound 3 crystalline form described hereinrefers to an amount sufficient to elicit a desired biological response,i.e., treating the condition. As will be appreciated by those ofordinary skill in this art, the effective amount of a crystalline formof Compound 3 described herein may vary depending on such factors as thedesired biological endpoint, the pharmacokinetics of the Compound 3crystalline form, the condition being treated, the mode ofadministration, and the age and health of the subject. An effectiveamount encompasses therapeutic and prophylactic treatment. For example,in treating cancer, an effective amount of a Compound 3 crystalline formdescribed herein may reduce the tumor burden or stop the growth orspread of a tumor. In treating macular degeneration, an effective amountof a Compound 3 crystalline form described herein may improve sight,reduce the risk of vision loss, or prevent central vision loss fromworsening.

A “therapeutically effective amount” of a Compound 3 crystalline formdescribed herein is an amount sufficient to provide a therapeuticbenefit in the treatment of a condition or to delay or minimize one ormore symptoms associated with the condition. A therapeutically effectiveamount of a Compound 3 crystalline form described herein means an amountof a crystalline form of Compound 3, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms, signs,or causes of the condition, and/or enhances the therapeutic efficacy ofanother therapeutic agent. In certain embodiments, a “therapeuticallyeffective amount” of crystalline form of Compound 3 or compositionthereof is the amount needed to inhibit angiogenesis in a subject.

A “prophylactically effective amount” of a Compound 3 crystalline formdescribed herein is an amount sufficient to prevent a condition, or oneor more symptoms associated with the condition or prevent itsrecurrence. A prophylactically effective amount of a Compound 3crystalline form described herein means an amount of a crystalline formof Compound 3, alone or in combination with other agents, which providesa prophylactic benefit in the prevention of the condition. The term“prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

As used herein, the term “growth factor” refers to a naturally occurringsubstance (e.g., a protein or a steroid hormone) capable of stimulatingcellular growth, proliferation, and/or cellular differentiation. Growthfactors may act as signaling molecules between cells and/or promote celldifferentiation and maturation.

A “proliferative disease” refers to a disease that occurs due toabnormal growth or extension by the multiplication of cells (Walker,Cambridge Dictionary of Biology; Cambridge University Press: Cambridge,UK, 1990). A proliferative disease may be associated with: 1) thepathological proliferation of normally quiescent cells; 2) thepathological migration of cells from their normal location (e.g.,metastasis of neoplastic cells); 3) the pathological expression ofproteolytic enzymes such as the matrix metalloproteinases (e.g.,collagenases, gelatinases, and elastases); or 4) the pathologicalangiogenesis as in proliferative retinopathy and tumor metastasis.Exemplary proliferative diseases include cancers (i.e., “malignantneoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, andautoimmune diseases.

As used herein, the term “angiogenesis” refers to the physiologicalprocess through which new blood vessels form from pre-existing vessels.Angiogenesis is distinct from vasculogenesis, which is the de novoformation of endothelial cells from mesoderm cell precursors. The firstvessels in a developing embryo form through vasculogenesis, after whichangiogenesis is responsible for most blood vessel growth during normalor abnormal development. Angiogenesis is a vital process in growth anddevelopment, as well as in wound healing and in the formation ofgranulation tissue. However, angiogenesis is also a fundamental step inthe transition of tumors from a benign state to a malignant one, leadingto the use of angiogenesis inhibitors in the treatment of cancer.Angiogenesis may be chemically stimulated by angiogenic proteins, suchas growth factors (e.g., VEGF).

The terms “neoplasm” and “tumor” are used herein interchangeably andrefer to an abnormal mass of tissue wherein the growth of the masssurpasses and is not coordinated with the growth of a normal tissue. Aneoplasm or tumor may be “benign” or “malignant,” depending on thefollowing characteristics: degree of cellular differentiation (includingmorphology and functionality), rate of growth, local invasion, andmetastasis. A “benign neoplasm” is generally well differentiated, hascharacteristically slower growth than a malignant neoplasm, and remainslocalized to the site of origin. In addition, a benign neoplasm does nothave the capacity to infiltrate, invade, or metastasize to distantsites. Exemplary benign neoplasms include, but are not limited to,lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheickeratoses, lentigos, and sebaceous hyperplasias. In some cases, certain“benign” tumors may later give rise to malignant neoplasms, which mayresult from additional genetic changes in a subpopulation of the tumor'sneoplastic cells, and these tumors are referred to as “pre-malignantneoplasms.” An exemplary pre-malignant neoplasm is a teratoma. Incontrast, a “malignant neoplasm” is generally poorly differentiated(anaplasia) and has characteristically rapid growth accompanied byprogressive infiltration, invasion, and destruction of the surroundingtissue. Furthermore, a malignant neoplasm generally has the capacity tometastasize to distant sites. The term “metastasis,” “metastatic,” or“metastasize” refers to the spread or migration of cancerous cells froma primary or original tumor to another organ or tissue and is typicallyidentifiable by the presence of a “secondary tumor” or “secondary cellmass” of the tissue type of the primary or original tumor and not ofthat of the organ or tissue in which the secondary (metastatic) tumor islocated. For example, a prostate cancer that has migrated to bone issaid to be metastasized prostate cancer and includes cancerous prostatecancer cells growing in bone tissue.

As used herein, the term “cancer” refers to a malignant neoplasm(Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins:Philadelphia, 1990). Exemplary cancers include, but are not limited to:acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer;angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma,hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliarycancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g.,adenocarcinoma of the breast, papillary carcinoma of the breast, mammarycancer, medullary carcinoma of the breast); brain cancer (e.g.,meningioma, glioblastomas, glioma (e.g., astrocytoma,oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor;cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma;chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer,rectal cancer, colorectal adenocarcinoma); connective tissue cancer;epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi'ssarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer(e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g.,adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing'ssarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma);familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g.,stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germcell cancer; head and neck cancer (e.g., head and neck squamous cellcarcinoma, oral cancer (e.g., oral squamous cell carcinoma), throatcancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngealcancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemiasuch as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL),acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphomasuch as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) andnon-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large celllymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., Waldenström's macroglobulinemia), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease);hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastictumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastomaa.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g.,hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g.,bronchogenic carcinoma, small cell lung cancer (SCLC), non-small celllung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS);mastocytosis (e.g., systemic mastocytosis); muscle cancer;myelodysplastic syndrome (MDS); mesothelioma; myeloproliferativedisorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis(ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF),chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML),chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES));neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreaticneuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g.,bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma;pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer(e.g., Paget's disease of the penis and scrotum); pinealoma; primitiveneuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplasticsyndromes; intraepithelial neoplasms; prostate cancer (e.g., prostateadenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer;skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g.,appendix cancer); soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous glandcarcinoma; small intestine cancer; sweat gland carcinoma; synovioma;testicular cancer (e.g., seminoma, testicular embryonal carcinoma);thyroid cancer (e.g., papillary carcinoma of the thyroid, papillarythyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer;vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).

As used herein, the term “inflammatory disease” or “inflammation” refersto a disease caused by, resulting from, or resulting in inflammation.The term “inflammatory disease” may also refer to a dysregulatedinflammatory reaction that causes an exaggerated response bymacrophages, granulocytes, and/or T-lymphocytes leading to abnormaltissue damage and/or cell death. An inflammatory disease can be eitheran acute or chronic inflammatory condition and can result frominfections or non-infectious causes. Inflammatory diseases include,without limitation, atherosclerosis, arteriosclerosis, autoimmunedisorders, multiple sclerosis, systemic lupus erythematosus, polymyalgiarheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis,bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoidarthritis, inflammatory arthritis, Sjogren's syndrome, giant cellarteritis, progressive systemic sclerosis (scleroderma), ankylosingspondylitis, polymyositis, dermatomyosifis, pemphigus, pemphigoid,diabetes (e.g., Type I), myasthenia gravis, Hashimoto's thyroditis,Graves' disease, Goodpasture's disease, mixed connective tissue disease,sclerosing cholangitis, inflammatory bowel disease, Crohn's disease,ulcerative colitis, pernicious anemia, inflammatory dermatoses, usualinterstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis,berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamativeinterstitial pneumonia, lymphoid interstitial pneumonia, giant cellinterstitial pneumonia, cellular interstitial pneumonia, extrinsicallergic alveolitis, Wegener's granulomatosis and related forms ofangiitis (temporal arteritis and polyarteritis nodosa), inflammatorydermatoses, hepatitis, delayed-type hypersensitivity reactions (e.g.,poison ivy dermatitis), pneumonia, respiratory tract inflammation, AdultRespiratory Distress Syndrome (ARDS), encephalitis, immediatehypersensitivity reactions, asthma, hayfever, allergies, acuteanaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis,cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury),reperfusion injury, allograft rejection, host-versus-graft rejection,appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis,cervicitis, cholangitis, chorioamnionitis, conjunctivitis,dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis,enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis,gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis,myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis,osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis,pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, rhinitis,salpingitis, sinusitis, stomatitis, synovitis, testitis, tonsillitis,urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis,vulvovaginitis, angitis, chronic bronchitis, osteomylitis, opticneuritis, temporal arteritis, transverse myelitis, necrotizingfascilitis, and necrotizing enterocolitis. Ocular inflammatory diseasesinclude, but are not limited to, allergy of the eye, uveitis (e.g.,anterior uveitis, intermediate uveitis, and posterior uveitis),conjunctivitis, panuveitis, cyclitis, scleritis, episcleritis, opticneuritis, retrobulbar optic neuritis, keratitis (e.g., immune keratitisand infectious keratitis), blepharitis, meibomian gland disease ordysfunction, corneal ulcer, conjunctival ulcer and symptoms caused bythem, ocular inflammatory diseases caused by ocular disorders, ocularinflammatory diseases caused by a physical injury, post-surgicalinflammation, and dry eye (e.g., dry eye syndrome).

As used herein, an “autoimmune disease” refers to a disease arising froman inappropriate immune response of the body of a subject againstsubstances and tissues normally present in the body. In other words, theimmune system mistakes some part of the body as a pathogen and attacksits own cells. This may be restricted to certain organs (e.g., inautoimmune thyroiditis) or involve a particular tissue in differentplaces (e.g., Goodpasture's disease which may affect the basementmembrane in both the lung and kidney). The treatment of autoimmunediseases is typically with immunosuppressants, e.g., medications thatdecrease the immune response. Exemplary autoimmune diseases include, butare not limited to, glomerulonephritis, Goodspasture's syndrome,necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemiclupus erythematosis, rheumatoid, arthritis, psoriatic arthritis,systemic lupus erythematosis, psoriasis, ulcerative colitis, systemicsclerosis, dermatomyositis/polymyositis, anti-phospholipid antibodysyndrome, scleroderma, perphigus vulgaris, ANCA-associated vasculitis(e.g., Wegener's granulomatosis, microscopic polyangiitis), urveitis,Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosingspondylitis, Lyme arthritis, GuillainBarre syndrome, Hashimoto'sthyroiditis, and cardiomyopathy.

The term “autoinflammatory disease” refers to a category of diseasesthat are similar but different from autoimmune diseases.Autoinflammatory and autoimmune diseases share common characteristics inthat both groups of disorders result from the immune system attacking asubject's own tissues and result in increased inflammation. Inautoinflammatory diseases, a subject's innate immune system causesinflammation for unknown reasons. The innate immune system reacts eventhough it has never encountered autoantibodies or antigens in thesubject. Autoinflammatory disorders are characterized by intenseepisodes of inflammation that result in such symptoms as fever, rash, orjoint swelling. These diseases also carry the risk of amyloidosis, apotentially fatal buildup of a blood protein in vital organs.Autoinflammatory diseases include, but are not limited to, familialMediterranean fever (FMF), neonatal onset multisystem inflammatorydisease (NOMID), tumor necrosis factor (TNF) receptor-associatedperiodic syndrome (TRAPS), deficiency of the interleukin-1 receptorantagonist (DIRA), and Behçet's disease.

The term “biological sample” refers to any sample including tissuesamples (such as tissue sections and needle biopsies of a tissue); cellsamples (e.g., cytological smears (such as Pap or blood smears) orsamples of cells obtained by microdissection); samples of wholeorganisms (such as samples of yeasts or bacteria); or cell fractions,fragments or organelles (such as obtained by lysing cells and separatingthe components thereof by centrifugation or otherwise). Other examplesof biological samples include blood, serum, urine, semen, fecal matter,cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus,biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy),nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccalswabs), or any material containing biomolecules that is derived from afirst biological sample. Biological samples also include thosebiological samples that are transgenic, such as transgenic oocyte, spermcell, blastocyst, embryo, fetus, donor cell, or cell nucleus.

The term “ocular disease” or “ocular disorder” refers to any eye diseaseand/or disorder. For example, ocular diseases can be disorders of theeyelid, lacrimal system and orbit, disorders of conjunctiva, disordersof sclera, cornea, iris and ciliary body, disorders of choroid,disorders of retina, glaucoma, disorders of optic nerve and visualpathways, ocular neovascularization diseases or disorders, ocularinflammatory diseases, or disorders of ocular muscles. Additionally,ocular disease can also refer to discomfort following injury, surgery,or laser treatment. Diseases and disorders of the eye or ocular diseasesinclude, but are not limited to, retinopathy, diabetic retinopathy,retinal vein occlusion, macular degeneration, age-related maculardegeneration, dry eye syndrome, blepharitis, inflammatory meibomiangland disease, uveitis, allergic conjunctivitis, glaucoma, macularedema, diabetic macular edema, cystoid macular edema, and rosacea (ofthe eye). Dry eye syndrome (DES), otherwise known askeratoconjunctivitis sicca (KCS), keratitis sicca, sicca syndrome, orxerophthalmia, is an eye disease caused by decreased tear production orincreased tear film evaporation commonly found in humans and someanimals.

The term “age-related macular degeneration” or “AMD” is an oculardisease which usually affects older adults and results in a loss ofvision in the center of the visual field (the macula) because of damageto the retina. It occurs in “dry” and “wet” forms. It is a major causeof blindness and visual impairment in older adults (>50 years).

Macular degeneration can make it difficult or impossible to read orrecognize faces, although enough peripheral vision remains to allowother activities of daily life. The macula is the central area of theretina, which provides the most detailed central vision. In the dry(nonexudative) form, cellular debris called drusen accumulate betweenthe retina and the choroid, and the retina can become detached. In thewet (exudative) form, which is more severe, blood vessels grow up fromthe choroid behind the retina, and the retina can also become detached.It can be treated with laser coagulation, and with medication that stopsand sometimes reverses the growth of blood vessels. Macular degenerationincludes some macular dystrophies affecting younger subjects as well asage-related macular degeneration (AMD or ARMD), which is more commonlyknown. AMD begins with characteristic yellow deposits (drusen) in themacula, between the retinal pigment epithelium and the underlyingchoroid. Most patients with these early changes (referred to asage-related maculopathy) have good vision. Patients with drusen can goon to develop advanced AMD. The risk is considerably higher when thedrusen are large and numerous and associated with disturbance in thepigmented cell layer under the macula. Recent research suggests thatlarge and soft drusen are related to elevated cholesterol deposits andmay respond to cholesterol-lowering agents.

The term “macular edema” refers to the ocular diseases cystoid macularedema (CME) or diabetic macular edema (DME). CME is an ocular diseasewhich affects the central retina or macula of the eye. When thiscondition is present, multiple cyst-like (cystoid) areas of fluid appearin the macula and cause retinal swelling or edema. CME may accompany avariety of diseases such as retinal vein occlusion, uveitis, and/ordiabetes. CME commonly occurs after cataract surgery. DME occurs whenblood vessels in the retina of patients with diabetes begin to leak intothe macula. These leaks cause the macula to thicken and swell,progressively distorting acute vision. While the swelling may not leadto blindness, the effect can cause a severe loss in central vision.

The term “glaucoma” refers to an ocular disease in which the optic nerveis damaged in a characteristic pattern. This can permanently damagevision in the affected eye and lead to blindness if left untreated. Itis normally associated with increased fluid pressure in the eye (aqueoushumor). The term ocular hypertension is used for patients withconsistently raised intraocular pressure (IOP) without any associatedoptic nerve damage. Conversely, the term normal tension or low tensionglaucoma is used for those with optic nerve damage and associated visualfield loss but normal or low IOP. The nerve damage involves loss ofretinal ganglion cells in a characteristic pattern. There are manydifferent subtypes of glaucoma, but they can all be considered to be atype of optic neuropathy. Raised intraocular pressure (e.g., above 21mmHg or 2.8 kPa) is the most important and only modifiable risk factorfor glaucoma. However, some may have high eye pressure for years andnever develop damage, while others can develop nerve damage at arelatively low pressure. Untreated glaucoma can lead to permanent damageof the optic nerve and resultant visual field loss, which over time canprogress to blindness.

The term “uveitis” refers to an inflammatory disease of the uvea, thevascular layer of the eye sandwiched between the retina and the white ofthe eye (sclera). The uvea extends toward the front of the eye andconsists of the iris, choroid layer and ciliary body. Uveitis includesanterior uveitis, intermediate uveitis, and posterior uveitis. A mostcommon type of uveitis is an inflammation of the iris called iritis(anterior uveitis). Uveitis may also occur at the posterior segment ofthe eye (e.g., at the choroid). Inflammation of the uvea can berecurring and can cause serious problems such as blindness if leftuntreated (accounts for 10% of blindness globally). Early diagnosis andtreatment are important to prevent the complications of uveitis.

The term “dry eye” or “dry eyes” refers to an ocular disease in whichthere are insufficient tears to lubricate and nourish the eye. Tears arenecessary for maintaining the health of the front surface of the eye andfor providing clear vision. Patients with dry eyes either do not produceenough tears or have a poor quality of tears. Dry eye is a common andoften chronic problem, particularly in older adults. With each blink ofthe eyelids, tears are spread across the front surface of the eye, knownas the cornea. Tears provide lubrication, reduce the risk of eyeinfection, wash away foreign matter in the eye, and keep the surface ofthe eyes smooth and clear. Excess tears in the eyes flow into smalldrainage ducts, in the inner corners of the eyelids, which drain in theback of the nose. Tears are produced by several glands (e.g., lacrimalgland) in and around the eyelids. Tear production tends to diminish withage, with various medical conditions, or as a side effect of certainmedicines. Environmental conditions such as wind and dry climates canalso affect tear volume by increasing tear evaporation. When the normalamount of tear production decreases or tears evaporate too quickly fromthe eyes, symptoms of dry eye can develop. The most common form of dryeyes is due to an inadequate amount of the water layer of tears. Thiscondition, called keratoconjunctivitis sicca (KCS), is also referred toas “dry eye syndrome.”

The term “diabetic retinopathy” refers to retinopathy (i.e., a diseaseof the retina) caused by complications of diabetes, which can eventuallylead to blindness. Diabetic retinopathy may cause no symptoms, mildvision problems, or even blindness. Diabetic retinopathy is the resultof microvascular retinal changes. Hyperglycemia-induced intramuralpericyte death and thickening of the basement membrane lead toincompetence of the vascular walls. These damages change the formationof the blood-retinal barrier and also make the retinal blood vesselsbecome more permeable. The pericyte death is caused when hyperglycemiapersistently activates protein kinase C-δ (PKC-δ, encoded by Prkcd) andp38 mitogen-activated protein kinase (MAPK) to increase the expressionof a previously unknown target of PKC-δ signaling, Src homology-2domain-containing phosphatase-1 (SHP-1), a protein tyrosine phosphatase.This signaling cascade leads to PDGF receptor-dephosphorylation and areduction in downstream signaling from this receptor, resulting inpericyte apoptosis. Small blood vessels, such as those in the eye, areespecially vulnerable to poor control over blood sugar. Anoveraccumulation of glucose and/or fructose damages the tiny bloodvessels in the retina. During the initial stage, called“nonproliferative diabetic retinopathy” (NPDR), most patients do notnotice any change in their vision. Early changes that are reversible anddo not threaten central vision are sometimes termed simplex retinopathyor background retinopathy. As the disease progresses, severenonproliferative diabetic retinopathy enters an advanced, “proliferativediabetic retinopathy” (PDR) stage when blood vessels proliferate. Thelack of oxygen in the retina causes fragile, new, blood vessels to growalong the retina and in the clear, gel-like vitreous humor that fillsthe inside of the eye, which may result in bleeding, cloudy vision,retina damage, or tractional retinal detachment.

The term “VEGF” is used interchangeably with vascular endothelial growthfactor herein. VEGF includes, but is not limited to, VEGF-relatedproteins such as placenta growth factor (PIGF), VEGF-A, VEGF-B, VEGF-C,VEGF-D, VEGF-E, and VEGF-F. The term VEGF also covers a number ofproteins from two families that result from alternate splicing of mRNAfrom a single, 8-exon, VEGF gene. The two different families arereferred to according to their terminal exon (exon 8) splice site—theproximal splice site (denoted VEGF_(xxx)) or distal splice site(VEGF_(xxxb)). In addition, alternate splicing of exon 6 and 7 alterstheir heparin-binding affinity, and amino acid number (in humans:VEGF₁₂₁, VEGF_(121b), VEGF₁₄₅, VEGF₁₆₅, VEGF_(165b), VEGF₁₈₉, VEGF₂₀₆;the rodent orthologs of these proteins contain one fewer amino acid).These domains have important functional consequences for the VEGF splicevariants, as the terminal (exon 8) splice site determines whether theproteins are pro-angiogenic (proximal splice site, expressed duringangiogenesis) or anti-angiogenic (distal splice site, expressed innormal tissues). In addition, inclusion or exclusion of exons 6 and 7mediate interactions with heparan sulfate proteoglycans (HSPGs) andneuropilin co-receptors on the cell surface, enhancing their ability tobind and activate the VEGF receptors (VEGFRs). The term “VEGF” alsoencompasses VEGF receptors. There are three main subtypes of VEGFR,numbered 1, 2 and 3. Also, they may be membrane-bound (mbVEGFR) orsoluble (sVEGFR), depending on alternative splicing.

The term “particle” refers to a small object, fragment, or piece of asubstance that may be a single element, inorganic material, organicmaterial, or mixture thereof. Examples of particles include polymericparticles, single-emulsion particles, double-emulsion particles,coacervates, liposomes, microparticles, nanoparticles, macroscopicparticles, pellets, crystals (e.g., crystalline forms of compounds oractive pharmaceutical agent), aggregates, composites, pulverized,milled, or otherwise disrupted matrices, and cross-linked protein orpolysaccharide particles, each of which have an average characteristicdimension of about less than about 1 mm and at least 1 nm, where thecharacteristic dimension, or “critical dimension,” of the particle isthe smallest cross-sectional dimension of the particle. A particle maybe composed of a single substance or multiple substances. In certainembodiments, the particle is not a viral particle. In other embodiments,the particle is not a liposome. In certain embodiments, the particle isnot a micelle. In certain embodiments, the particle is substantiallysolid throughout. In certain embodiments, the particle is ananoparticle. In certain embodiments, the particle is a microparticle.

The term “nanoparticle” refers to a particle having a characteristicdimension of less than about 1 micrometer and at least about 1nanometer, where the characteristic dimension of the particle is thesmallest cross-sectional dimension of the particle. A crystallinenanoparticle is referred to as a “nanocrystal.”

The term “microparticle” refers to a particle having a characteristicdimension of less than about 1 millimeter and at least about 1micrometer, where the characteristic dimension of the particle is thesmallest cross-sectional dimension of the particle.

The term “nanostructure” refers to a structure having at least oneregion or characteristic dimension with a dimension of less than about1000 nm, e.g., less than about 300 nm, less than about 200 nm, less thanabout 100 nm, or less than about 50 nm. Typically, the region orcharacteristic dimension will be along the smallest axis of thestructure. Examples of such structures include nanowires, nanorods,nanotubes, branched nanocrystals, nanotetrapods, tripods, bipods,nanocrystals, nanodots, quantum dots, nanoparticles, branched tetrapods(e.g., inorganic dendrimers), and the like. Nanostructures can besubstantially homogeneous in material properties, or in certainembodiments can be heterogeneous (e.g. heterostructures). Nanostructurescan be, e.g., substantially crystalline, substantially monocrystalline,polycrystalline, amorphous, or a combination thereof. In one aspect,each of the three dimensions of the nanostructure has a dimension ofless than about 1000 nm, e.g., or even less than about 300 nm, less thanabout 200 nm, less than about 100 nm, or less than about 50 nm.Nanostructures can comprise one or more surface ligands (e.g.,surfactants).

The terms “crystalline” or “substantially crystalline”, when used withrespect to nanostructures, refer to the fact that the nanostructurestypically exhibit long-range ordering across one or more dimensions ofthe structure. It will be understood by one of skill in the art that theterm “long range ordering” will depend on the absolute size of thespecific nanostructures, as ordering for a single crystal cannot extendbeyond the boundaries of the crystal. In this case, “long-rangeordering” will mean substantial order across at least the majority ofthe dimension of the nanostructure. In some instances, a nanostructurecan bear an oxide or other coating, or can be comprised of a core and atleast one shell. In such instances it will be appreciated that theoxide, shell(s), or other coating need not exhibit such ordering (e.g.it can be amorphous, polycrystalline, or otherwise). In such instances,the phrase “crystalline,” “substantially crystalline,” “substantiallymonocrystalline,” or “monocrystalline” refers to the central core of thenanostructure (excluding the coating layers or shells). The terms“crystalline” or “substantially crystalline” as used herein are intendedto also encompass structures comprising various defects, stackingfaults, atomic substitutions, and the like, as long as the structureexhibits substantial long range ordering (e.g., order over at leastabout 80% of the length of at least one axis of the nanostructure or itscore). In addition, it will be appreciated that the interface between acore and the outside of a nanostructure or between a core and anadjacent shell or between a shell and a second adjacent shell maycontain non-crystalline regions and may even be amorphous. This does notprevent the nanostructure from being crystalline or substantiallycrystalline as defined herein. The term “monocrystalline” when used withrespect to a nanostructure indicates that the nanostructure issubstantially crystalline and comprises substantially a single crystal.When used with respect to a nanostructure heterostructure comprising acore and one or more shells, “monocrystalline” indicates that the coreis substantially crystalline and comprises substantially a singlecrystal. When not used with respect to a nanostructure, the term“monocrystalline” to materials that are composed of substantially asingle crystallite of substantially the same size and orientation.

“Nanocrystal” is a nanostructure that is substantially monocrystalline.A nanocrystal thus has at least one region or characteristic dimensionwith a dimension of less than about 1000 nm, e.g., less than about 300nm less than about 200 nm, less than about 100 nm, or less than about 50nm. Typically, the region or characteristic dimension will be along thesmallest axis of the structure. Examples of such structures includenanowires, nanorods, nanotubes, branched nanowires, nanotetrapods,nanotripods, nanobipods, nanocrystals, nanodots, quantum dots,nanoparticles, nanoribbons, and the like. Nanostructures can besubstantially homogeneous in material properties, or in certainembodiments can be heterogeneous (e.g. heterostructures). Optionally, ananocrystal can comprise one or more surface ligands (e.g.,surfactants). The nanocrystal is optionally substantially single crystalin structure (a “single crystal nanostructure” or a “monocrystallinenanostructure”). While nanostructures for use in the present inventioncan be fabricated from essentially any convenient material or material,preferably the nanostructure is prepared from an inorganic material,e.g., an inorganic conductive or semiconductive material. A conductiveor semiconductive nanostructure often displays 1-dimensional quantumconfinement, e.g., an electron can often travel along only one dimensionof the structure. Nanocrystals can be substantially homogeneous inmaterial properties, or in certain embodiments can be heterogeneous(e.g. heterostructures). The term “nanocrystal” is intended to encompasssubstantially monocrystalline nanostructures comprising various defects,stacking faults, atomic substitutions, and the like, as well assubstantially monocrystalline nanostructures without such defects,faults, or substitutions. In the case of nanocrystal heterostructurescomprising a core and one or more shells, the core of the nanocrystal istypically substantially monocrystalline, but the shell(s) need not be.The nanocrystals can be fabricated from essentially any convenientmaterial or materials.

The term “polycrystalline” refers to materials that are composed of manycrystallites of varying size and orientation. When used with respect tonanostructures, the term “polycrystalline” refers to a crystallinenanostructure that is not monocrystalline.

A “biocompatible” material refers to a material that does not typicallyinduce an adverse response when inserted or injected into a subject. Theadverse response includes significant inflammation and/or acuterejection of the material by the immune system of the subject, forinstance, via a T-cell-mediated response. It is recognized that“biocompatibility” is a relative term and that some degree of immuneresponse is to be expected even for materials that are highly compatiblewith living tissues of the subject. However, as used herein,“biocompatibility” refers to the acute rejection of a material by atleast a portion of the immune system, i.e., a material that lacksbiocompatibility (i.e. being non-biocompatible) in a subject provokes animmune response in the subject that is severe enough such that therejection of the material by the immune system cannot be adequatelycontrolled and often is of a degree such that the material must beremoved from the subject in order for the subject to be as well as itwas before the non-biocompatible material was introduced into thesubject. One test to determine biocompatibility of a material is toexpose the material to cells (e.g., fibroblasts or epithelial cells) invitro; the material is considered biocompatible if it does not result insignificant cell death at moderate concentrations, e.g., atconcentrations of about 50 micrograms/10⁶ cells. In certain embodiments,there is no significant cell death if less than about 20% of the cellsare dead, even if phagocytosed or otherwise uptaken by the cells. Insome embodiments, a material is biocompatible if contacting it withcells in vitro results in less than 20% cell death and if theadministration of the material in vivo does not induce unwantedinflammation or other adverse responses. In certain embodiments, abiocompatible material is biodegradable. A non-limiting example ofbiocompatible materials is biocompatible polymers (includingbiocompatible copolymers).

A “biodegradable” material refers to a material that is able to degradechemically and/or biologically (e.g., by hydrolysis or enzymaticactivity), within a physiological environment, such as within the bodyor when introduced to cells. For instance, the material may be one thathydrolyzes spontaneously upon exposure to water (e.g., within a subject)and/or may degrade upon exposure to heat (e.g., at temperatures of about37° C.). Degradation of a material may occur at varying rates, dependingon the material used. For example, the half-life of the material (thetime at which 50% of the material is degraded into smaller components)may be on the order of days, weeks, months, or years. The material maybe biologically degraded, e.g., by enzymatic activity or cellularmachinery, for example, through exposure to a lysozyme. In someembodiments, the material may be broken down into smaller componentsthat cells can either reuse or dispose of without significant toxiceffect on the cells (e.g., fewer than about 20% of the cells are killedwhen the components are added to cells in vitro). Non-limiting examplesof biodegradable materials are biodegradable polymers (includingbiodegradable copolymers). Examples of biodegradable polymers include,but are not limited to, poly(ethylene glycol)-poly(propyleneoxide)-poly(ethylene glycol) triblock copolymers, poly(vinyl alcohol)(PVA), poly(lactide) (or poly(lactic acid)), poly(glycolide) (orpoly(glycolic acid)), poly(orthoesters), poly(caprolactones),polylysine, poly(ethylene imine), poly(acrylic acid), poly(urethanes),poly(anhydrides), poly(esters), poly(trimethylene carbonate),poly(ethyleneimine), poly(acrylic acid), poly(urethane), poly(beta aminoesters), and copolymers thereof (e.g., poly(lactide-co-glycolide)(PLGA)).

As used herein, the terms “pharmaceutical composition” and “formulation”are used interchangeably.

As used herein, the terms “pharmaceutical agent” and “drug” are usedinterchangeably.

DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention provides crystalline forms of the compound7-(3-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinazolin-7-yloxy)propyl)-2-oxa-7-azaspiro[3.5]nonane,referred to herein as Compound 3 and shown below:

In particular embodiments, the crystalline form is crystalline Form A,which has an X-Ray Powder Diffraction (XRPD) pattern with peaks at about6.11, 9.63, 16.41, 18.60, 20.36 and 23.01±0.3 degrees two theta or14.45, 9.17, 5.40, 4.77, 4.36 and 3.86±0.3 Å in d-spacing. In furtherembodiments, crystalline Form A further has XRPD peaks at about 11.46,12.26, 18.16, 19.51, 21.12 and 25.71±0.3 degrees two theta or 7.71,7.22, 4.88, 4.55, 4.20 and 3.46±0.3 Å in d-spacing. In furtherembodiments, crystalline Form A further has XRPD peaks at about 11.10,15.66, 17.54, 22.31, 24.79 and 28.90±0.3 degrees two theta or 7.96,5.65, 5.05, 3.98, 3.59 and 3.09±0.3 Å in d-spacing. In still furtherembodiments, crystalline Form A has an XRPD pattern with peaks at about6.11, 9.63, 11.10, 11.46, 12.26, 15.66, 16.41, 17.54, 18.16, 18.60,19.51, 20.36, 21.12, 22.31, 23.01, 24.79, 25.71 and 28.90±0.3 degreestwo theta or 14.45, 9.17, 7.96, 7.71, 7.22, 5.65, 5.40, 5.05, 4.88,4.77, 4.55, 4.36, 4.20, 3.98, 3.86, 3.59, 3.46 and 3.09±0.3 Å ind-spacing.

In other particular embodiments, the crystalline form is crystallineForm B, which has an XRPD pattern with peaks at about 7.70, 13.53,17.27, 18.44, 19.73, 23.10 and 26.07±0.3 degrees two theta or 11.47,6.54, 5.13, 4.81, 4.50, 3.85 and 3.41±0.3 Å in d-spacing. In furtherembodiments, crystalline Form B further has XRPD peaks at about 9.87,12.88, 14.40, 15.45, 21.14 and 26.84±0.3 degrees two theta or 8.96,6.87, 6.14, 5.73, 4.20 and 3.32±0.3 Å in d-spacing. In furtherembodiments, crystalline Form B further has XRPD peaks at about 10.69,16.42, 18.90, 22.56, and 29.12±0.3 degrees two theta or 8.27, 5.39,4.69, 3.94 and 3.06±0.3 Å in d-spacing. In still further embodiments,crystalline Form B has an XRPD pattern with peaks at about 7.70, 9.87,10.69, 12.88, 13.53, 14.40, 15.45, 16.42, 17.27, 18.44, 18.90, 19.73,21.14, 22.56, 23.10, 26.07, 26.84 and 29.12±0.3 degrees two theta or11.47, 8.96, 8.27, 6.87, 6.54, 6.14, 5.73, 5.39, 5.13, 4.81, 4.69, 4.50,4.20, 3.94, 3.85, 3.41, 3.32 and 3.06±0.3 Å in d-spacing.

In one aspect, the present invention relates to a compound having theformula

in crystalline Form A.

In another aspect, the present invention relates to a crystalline formof a compound having the formula

wherein said crystalline form is crystalline Form A having an X-raypowder diffraction (XRPD) pattern with peaks at about 6.11, 9.63, 16.41,18.60, 20.36 and 23.01±0.3 degrees two theta, or 14.45, 9.17, 5.40,4.77, 4.36 and 3.86±0.3 Å in d-spacing.

In another embodiment, the present invention relates to a compoundhaving the formula

in crystalline Form B.

In another embodiment, the present invention relates to a crystallineform of a compound having the formula

wherein said crystalline form is crystalline Form B having an X-RayPowder Diffraction (XRPD) pattern with peaks at about 7.70, 13.53,17.27, 18.44, 19.73, 23.10 and 26.07±0.3 degrees two theta or 11.47,6.54, 5.13, 4.81, 4.50, 3.85 and 3.41±0.3 Å in d-spacing.

The present invention also relates to a process for preparing acrystalline form of Compound 3. In certain embodiments, the presentinvention relates to a method for preparing crystalline Form A ofCompound 3. In additional embodiments, the method of preparingcrystalline Form A comprises wet-milling a slurry comprising anamorphous form of Compound 3 and a non-ionic surfactant to obtainnanoparticles of the compound. In further embodiments, the resultingnanoparticles of crystalline Form A have an XRPD pattern with peaks atabout 6.11, 9.63, 16.41, 18.60, 20.36 and 23.01±0.3 degrees two theta or14.45, 9.17, 5.40, 4.77, 4.36 and 3.86±0.3 Å in d-spacing. In stillfurther embodiments, the resulting nanoparticles of crystalline Form Afurther have an XRPD pattern with peaks at about 11.46, 12.26, 18.16,19.51, 21.12 and 25.71±0.3 degrees two theta or 7.71, 7.22, 4.88, 4.55,4.20 and 3.46±0.3 Å in d-spacing, or at about 11.10, 15.66, 17.54,22.31, 24.79 and 28.9±0.3 degrees two theta or 7.96, 5.65, 5.05, 3.98,3.59 and 3.09±0.3 Å in d-spacing, or both. In additional embodiments,the resulting nanoparticles of crystalline Form A have an XRPD patternwith peaks at about 6.11, 9.63, 11.10, 11.46, 12.26, 15.66, 16.41,17.54, 18.16, 18.60, 19.51, 20.36, 21.12, 22.31, 23.01, 24.79, 25.71 and28.9±0.3 degrees two theta or 14.45, 9.17, 7.96, 7.71, 7.22, 5.65, 5.40,5.05, 4.88, 4.77, 4.55, 4.36, 4.20, 3.98, 3.86, 3.59, 3.46 and 3.09±0.3Å in d-spacing.

In other embodiments, the present invention relates to a method forpreparing crystalline Form B of Compound 3. In certain embodiments, themethod of preparing crystalline Form B comprises of crystallizing theamorphous form of Compound 3 from a solvent mixture comprising water andacetone. In particular embodiments, the method of preparing crystallineForm B utilizes a solvent mixture consisting of a 4:1 acetone:watermixture. In other embodiments, the method of preparing crystalline FormB further comprises heating the solvent mixture to dissolve the compoundand/or cooling the solvent mixture to allow crystal formation. In someembodiments, the resulting crystalline Form B has an XRPD pattern withpeaks at about 7.7, 13.53, 17.27, 18.44, 19.73, 23.1 and 26.07±0.3degrees two theta or 11.47, 6.54, 5.13, 4.81, 4.5, 3.85 and 3.41±0.3 Åin d-spacing. In additional embodiments, the resulting crystalline FormB further have an XRPD pattern with peaks at about 9.87, 12.88, 14.4,15.45, 21.14 and 26.84±0.3 degrees two theta or 8.96, 6.87, 6.14, 5.73,4.2 and 3.32±0.3 Å in d-spacing, or at about 10.69, 16.42, 18.9, 22.56and 29.12±0.3 degrees two theta or 8.27, 5.39, 4.69, 3.94 and 3.06±0.3 Åin d-spacing, or both. In additional embodiments, the resultingcrystalline Form B have an XRPD pattern with peaks at about 7.7, 9.87,10.69, 12.88, 13.53, 14.4, 15.45, 16.42, 17.27, 18.44, 18.9, 19.73,21.14, 22.56, 23.1, 26.07, 26.84 and 29.12±0.3 degrees two theta or11.47, 8.96, 8.27, 6.87, 6.54, 6.14, 5.73, 5.39, 5.13, 4.81, 4.69, 4.5,4.2, 3.94, 3.85, 3.41, 3.32 and 3.06±0.3 Å in d-spacing.

Also provided are methods of using the crystalline forms of Compound 3to treat diseases, including proliferative diseases, ocular diseases,dermatological diseases, inflammatory diseases, autoimmune diseases,auto-inflammatory diseases, and metabolic diseases. The presentinvention further provides methods of using crystalline Form A orcrystalline Form B of Compound 3 as therapeutics, e.g., in the treatmentand/or prevention of diseases associated with abnormal angiogenesisand/or aberrant signaling of a growth factor activity (e.g., vascularendothelial growth factor (VEGF) or angiogenesis. In certainembodiments, the disease being treated and/or prevented usingcrystalline Form A or crystalline Form B of Compound 3, pharmaceuticalcompositions, kits, uses, and methods include proliferative diseases(e.g., cancers, benign neoplasms, diseases associated with angiogenesis,inflammatory diseases, autoimmune diseases) and ocular diseases (e.g.,macular degeneration, glaucoma, diabetic retinopathy, retinoblastoma,edema, macular edema, corneal neovascularization, uveitis, dry eye,blepharitis, and post-surgical inflammation).

In certain embodiments, the crystalline forms of the invention aremonocrystalline. In certain embodiments, the compounds of the inventionare polycrystalline.

The crystalline forms of the invention may also have a relatively lowaqueous solubility (i.e., a solubility in water, optionally with one ormore buffers). For example, the crystalline forms of Compound 3 may havean aqueous solubility of less than about or equal to about 3 mg/mL, lessthan about 1 mg/mL, less than about 0.3 mg/mL, less than about 0.1mg/mL, less than about 0.03 mg/mL, less than about 0.01 mg/mL, less thanabout 1 μg/mL, less than about 0.1 μg/mL, less than about 0.01 μg/mL,less than about 1 ng/mL, less than about 0.1 ng/mL, or less than about0.01 ng/mL at 25° C. In some embodiments, the crystalline forms ofCompound 3 have an aqueous solubility of at least about 1 μg/mL, atleast about 10 μg/mL, at least about 0.1 ng/mL, at least about 1 ng/mL,at least about 10 ng/mL, at least about 0.1 μg/mL, at least about 1μg/mL, at least about 3 μg/mL, at least about 0.01 mg/mL, at least about0.03 mg/mL, at least about 0.1 mg/mL, at least about 0.3 mg/mL, at leastabout 1.0 mg/mL, or at least about 3 mg/mL at 25° C. Combinations of theabove-noted ranges are possible (e.g., an aqueous solubility of at leastabout 10 μg/mL and less than about 1 mg/mL). Other ranges are alsopossible. The crystalline forms of Compound 3 may have these or otherranges of aqueous solubilities at any point throughout the pH range(e.g., at about pH 7 or from pH 1 to pH 14).

The crystalline forms of Compound 3 may be suitable for being processedinto mucus-penetrating pharmaceutical compositions (e.g., particles orcrystals). In certain embodiments, the crystalline forms of Compound 3are suitable for milling (e.g., nano-milling). In certain embodiments,the crystalline forms of Compound 3 are suitable for precipitation(e.g., microprecipitation, nanoprecipitation, crystallization, orcontrolled crystallization). In certain embodiments, the crystallineforms of Compound 3 are suitable for emulsification. In certainembodiments, the crystalline forms of Compound 3 are suitable forfreeze-drying.

Compound 3 can be prepared using any suitable method. In certainembodiments, Compound 3 can be prepared using Method A as shown inScheme 1:

In certain embodiments, Compound 3 can also be prepared using Method Bas shown in Scheme 2:

Pharmaceutical Compositions, Kits, and Methods of Uses andAdministration

The present invention provides pharmaceutical compositions comprisingcrystalline Form A of Compound 3, and optionally a pharmaceuticallyacceptable excipient, or crystalline Form B of Compound 3, andoptionally a pharmaceutically acceptable excipient. In certainembodiments, a compound described herein is provided in an effectiveamount in the pharmaceutical composition. In certain embodiments, theeffective amount is a therapeutically effective amount. In certainembodiments, the effective amount is a prophylactically effectiveamount. In certain embodiments, the effective amount is an amounteffective for treating and/or preventing a disease. In certainembodiments, the effective amount is an amount effective for treating adisease. In certain embodiments, the effective amount is an amounteffective for treating and/or preventing a disease associated withaberrant signaling of a growth factor. In certain embodiments, theeffective amount is an amount effective for treating a diseaseassociated with aberrant signaling of a growth factor. In certainembodiments, the effective amount is an amount effective for treatingand/or preventing a disease associated with aberrant signaling ofvascular endothelial growth factor (VEGF). In certain embodiments, theeffective amount is an amount effective to treat and/or prevent adisease associated with abnormal angiogenesis, such as cancer, benignneoplasm, atherosclerosis, hypertension, inflammatory disease,rheumatoid arthritis, macular degeneration, choroidalneovascularization, retinal neovascularization, and diabeticretinopathy. In certain embodiments, the effective amount is an amounteffective to treat cancer (e.g., an ocular cancer). In certainembodiments, the effective amount is an amount effective to treatmacular degeneration.

An effective amount of Compound 3 crystalline form of the invention mayvary from about 0.001 mg/kg to about 1000 mg/kg in one or more doseadministrations for one or several days (depending on the mode ofadministration). In certain embodiments, the effective amount per dosevaries from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kgto about 750 mg/kg, from about 0.1 mg/kg to about 500 mg/kg, from about1.0 mg/kg to about 250 mg/kg, and from about 10.0 mg/kg to about 150mg/kg.

An effective amount of Compound 3 crystalline form of the invention mayinhibit abnormal angiogenesis and/or aberrant signaling of a growthfactor by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, or at least about 90%. An effective amount of aCompound 3 of the invention may inhibit abnormal angiogenesis and/oraberrant signaling of a growth factor by less than about 90%, less thanabout 80%, less than about 70%, less than about 60%, less than about50%, less than about 40%, less than about 30%, less than about 20%, orless than about 10%. Combinations of the ranges described herein (e.g.,at least 20% and less than 50%) are also within the scope of theinvention. In certain embodiments, an effective amount of a Compound 3of the invention inhibits abnormal angiogenesis and/or aberrantsignaling of a growth factor by a percentage or a range of percentagedescribed herein, compared to normal angiogenesis and/or signaling.

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing a crystalline form of Compound 3described herein (i.e., the “active ingredient”) into association with acarrier or excipient, and/or one or more other accessory ingredients,and then, if necessary and/or desirable, shaping, and/or packaging theproduct into a desired single- or multi-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.The composition may comprise between 0.001% and 100% (w/w) activeingredient.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose, and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, and mixtures thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g., acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays(e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminumsilicate)), long chain amino acid derivatives, high molecular weightalcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.,carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate (TWEEN® 20), polyoxyethylene sorbitan (TWEEN® 60),polyoxyethylene sorbitan monooleate (TWEEN® 80), sorbitan monopalmitate(SPAN® 40), sorbitan monostearate (SPAN® 60), sorbitan tristearate(SPAN® 65), glyceryl monooleate, sorbitan monooleate (SPAN® 80),polyoxyethylene esters (e.g., polyoxyethylene monostearate (MYRJ® 45),polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., CREMOPHOR®),polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (BRIJ® 30)),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLURONIC® F-68, Poloxamer P-188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, and/or mixtures thereof.

Exemplary binding agents include starch (e.g., cornstarch and starchpaste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g., acacia, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapol husks, carboxymethylcellulose,methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate(VEEGUM®), and larch arabogalactan), alginates, polyethylene oxide,polyethylene glycol, inorganic calcium salts, silicic acid,polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, antiprotozoanpreservatives, alcohol preservatives, acidic preservatives, and otherpreservatives. In certain embodiments, the preservative is anantioxidant. In other embodiments, the preservative is a chelatingagent.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT®Plus, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE®,KATHON®, and EUXYL®.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, and mixtures thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, chamomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary synthetic oils include, but are not limitedto, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixturesthereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredients,the liquid dosage forms may comprise inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents. Incertain embodiments for parenteral administration, the conjugates of theinvention are mixed with solubilizing agents such as CREMOPHOR®,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and mixtures thereof.

A pharmaceutical composition of the invention can be formulated foradministration by injection in any acceptable form, includingintravenous, intraperitoneal, intramuscular, subcutaneous, parenteral,epidural, or intraocular. Injectable preparations, for example, sterileinjectable aqueous or oleaginous suspensions can be formulated accordingto the known art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation can be a sterileinjectable solution, suspension, or emulsion in a nontoxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed including synthetic mono- or di-glycerides. Inaddition, fatty acids such as oleic acid are used in the preparation ofinjectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use. The formulation can also be prepared under asepticconditions or sterilized with heat or irradiation.

An injectable formulation or pharmaceutical composition of the inventioncan also be formulated for ophthalmic administration by injection in anyacceptable form, including intravitreal, perocular, intrastromal,intracameral, sub-retinal, conjunctival, subconjunctival, sub-tenon(e.g., anterior or posterior), circumcorneal, scleral, episcleral,posterior juxtascleral, peri-bulbar, retro-bulbar, suprachorodial, andtear duct. A pharmaceutical composition of the invention may also beformulated for ophthalmic administration by implant or the use ofreservoirs (e.g., biodegradable delivery system, non-biodegradabledelivery system and other implanted extended or slow release device orformulation).

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by formulating a Compound 3crystalline form of this invention with suitable non-irritatingexcipients or carriers such as cocoa butter, polyethylene glycol, or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or (a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, (c) humectants such as glycerol, (d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, (e) solutionretarding agents such as paraffin, (f) absorption accelerators such asquaternary ammonium compounds, (g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolinand bentonite clay, and (i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may include a buffering agent.

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the art of pharmacology. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of encapsulating compositions which can be used includepolymeric substances and waxes. Solid compositions of a similar type canbe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugar as well as high molecularweight polyethylene glycols and the like.

The active ingredient can be in a micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings, and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient can be admixed with at least oneinert diluent such as sucrose, lactose, or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of encapsulating agents which can be usedinclude polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a compoundof this invention may include ointments, pastes, creams, lotions, gels,powders, solutions, suspensions, sprays, inhalants, and/or patches.Generally, the active ingredient is admixed under sterile conditionswith a pharmaceutically acceptable carrier or excipient and/or anyneeded preservatives and/or buffers as can be required. Additionally,the present invention contemplates the use of transdermal patches, whichoften have the added advantage of providing controlled delivery of anactive ingredient to the body. Such dosage forms can be prepared, forexample, by dissolving and/or dispensing the active ingredient in theproper medium. Alternatively or additionally, the rate can be controlledby either providing a rate controlling membrane and/or by dispersing theactive ingredient in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionscan be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof.Alternatively or additionally, conventional syringes can be used in theclassical mantoux method of intradermal administration. Jet injectiondevices which deliver liquid vaccines to the dermis via a liquid jetinjector and/or via a needle which pierces the stratum corneum andproduces a jet which reaches the dermis are suitable. Jet injectiondevices are described, for example, in U.S. Pat. Nos. 5,480,381;5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627;5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; andPCT publications WO 97/37705 and WO 97/13537. Ballistic powder/particledelivery devices which use compressed gas to accelerate the compound inpowder form through the outer layers of the skin to the dermis aresuitable.

Formulations suitable for topical administration (including ocular ordermal) include, but are not limited to, liquid and/or semi-liquidpreparations such as liniments, lotions, oil-in-water and/orwater-in-oil emulsions such as creams, ointments, and/or pastes, and/orsolutions and/or suspensions. Topically administrable formulations may,for example, comprise from about 0.001% to about 50% (w/w) activeingredient, although the concentration of the active ingredient can beas high as the solubility limit of the active ingredient in the solvent.Formulations for topical administration may further comprise one or moreof the additional ingredients described herein. In one aspect, thepresent invention relates to formulations or pharmaceutical compositionssuitable for topical administration comprising crystalline Form A ofCompound 3 or crystalline Form B of Compound 3.

A pharmaceutical composition of the invention can be prepared, packaged,and/or sold in a formulation suitable for pulmonary administration. Sucha formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 toabout 7 microns, or from about 1 to about 6 microns. Such compositionsare conveniently in the form of dry powders for administration using adevice comprising a dry powder reservoir to which a stream of propellantcan be directed to disperse the powder and/or using a self-propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5nanometers and at least 95% of the particles by number have a diameterless than 20 microns. Alternatively, at least 95% of the particles byweight have a diameter greater than 1 nanometer and at least 90% of theparticles by number have a diameter less than 15 microns. Dry powdercompositions may include a solid fine powder diluent such as sugar andare conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.001 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may provide the active ingredient in the form of droplets of asolution and/or suspension. Such formulations can be prepared, packaged,and/or sold as aqueous and/or dilute alcoholic solutions and/orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization and/oratomization device. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, and/or a preservative such as methylhydroxybenzoate. Thedroplets provided by this route of administration may have an averagediameter in the range from about 0.01 to about 200 microns. Alternately,formulations for pulmonary administration may comprise a powder and/oran aerosolized and/or atomized solution and/or suspension comprising theactive ingredient. Such powdered, aerosolized, and/or atomizedformulations, when dispersed, may have an average particle and/ordroplet size in the range from about 0.01 to about 200 microns, and mayfurther comprise one or more of the additional ingredients describedherein.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition of theinvention. Another formulation suitable for intranasal administration isa coarse powder comprising the active ingredient and having an averageparticle from about 0.2 to 500 micrometers. Such a formulation isadministered by rapid inhalation through the nasal passage from acontainer of the powder held close to the nares. Formulations for nasaladministration may, for example, comprise from about as little as 0.001%(w/w) to as much as 100% (w/w) of the active ingredient, and maycomprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,and/or sold in a formulation for oral administration. Such formulationsmay, for example, be in the form of tablets and/or lozenges made usingconventional methods, and may contain, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable and/ordegradable composition and, optionally, one or more of the additionalingredients described herein.

Formulations described herein may also be delivered via buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and maycontain, for example, 0.001 to 50% (w/w) active ingredient, the balancecomprising an orally dissolvable and/or degradable composition and,optionally, one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,and/or sold in a formulation for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.001/10.0% (w/w) solution and/or suspension of theactive ingredient in an aqueous or oily liquid carrier or excipient.Such drops may further comprise buffering agents, salts, and/or one ormore other of the additional ingredients described herein. Otherophthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form and/or ina liposomal preparation.

A pharmaceutical composition of the invention may also be formulated foradministration by the ophthalmic mucous membrane route, such as, forexample, eye drops, ointments, or gels. These formulations may beprepared by conventional means, and, if desired, the subjectcompositions may be mixed with any conventional additive, such as abuffering or pH-adjusting agent, tonicity adjusting agents, viscositymodifiers, suspension stabilizers, preservatives, and otherpharmaceutical excipients. In addition, in certain embodiments, subjectcompositions described herein may be lyophilized or subjected to anotherappropriate drying technique such as spray drying. Ear drops are alsocontemplated as being within the scope of this invention.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.

Compositions provided herein are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular subject or organism will dependupon a variety of factors including the disease being treated and theseverity of the disorder; the activity of the specific active ingredientemployed; the specific composition employed; the age, body weight,general health, sex, and diet of the subject; the time ofadministration, route of administration, and rate of excretion of thespecific active ingredient employed; the duration of the treatment;drugs used in combination or coincidental with the specific activeingredient employed; and like factors well known in the medical arts.

The compositions provided herein can be administered by any route,including enteral (e.g., oral), parenteral, injection, intraocular,intravenous, intramuscular, intra-arterial, intramedullary, intrathecal,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (including dermal or ocular, suchas by powders, ointments, creams, and/or drops), mucosal, nasal, bucal,sublingual; by intratracheal instillation, bronchial instillation,and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.Specifically contemplated routes are oral administration, injections,including intravenous administration (e.g., systemic intravenousinjection) and intraocular administration, regional administration viablood and/or lymph supply, and/or direct administration to an affectedsite including topical administration (e.g., dermal and/or ocular). Ingeneral, the most appropriate route of administration will depend upon avariety of factors including the nature of the agent (e.g., itsstability in the environment of the gastrointestinal tract), and/or thecondition of the subject (e.g., whether the subject is able to tolerateoral administration). In certain embodiments, the compound orpharmaceutical composition of the invention is suitable foradministration to the eye of a subject. In another embodiment, thecompound or pharmaceutical composition of a crystalline form of Compound3 is suitable for topical administration to the eye of a subject.

The exact amount of a crystalline form of Compound 3 of the inventionrequired to achieve an effective amount will vary from subject tosubject, depending, for example, on species, age, and general conditionof a subject, severity of the side effects or disorder, mode ofadministration, and the like. The desired dosage can be delivered threetimes a day, two times a day, once a day, every other day, every thirdday, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage can be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

In certain embodiments, an effective amount of a Compound 3 crystallineform of the invention for administration one or more times a day to a 70kg adult human may comprise about 0.0001 mg to about 3000 mg, about0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mgto about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100mg, about 10 mg to about 1000 mg, about 10 mg to about 100 mg, or about100 mg to about 1000 mg, of Compound 3 per unit dosage form.

In certain embodiments, the Compound 3 crystalline forms describedherein may be at dosage levels sufficient to deliver from about 0.001mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 500 mg/kg,preferably from about 0.1 mg/kg to about 400 mg/kg, preferably fromabout 0.5 mg/kg to about 300 mg/kg, from about 0.01 mg/kg to about 100mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic and/or prophylacticeffect.

It will be appreciated that dose ranges as described herein provideguidance for the administration of provided pharmaceutical compositionsto an adult. The amount to be administered to, for example, a child oran adolescent can be determined by a medical practitioner or personskilled in the art and can be lower or the same as that administered toan adult.

It will be also appreciated that a crystalline form of Compound 3 orcomposition thereof, as described herein, can be administered incombination with one or more additional pharmaceutical agents (e.g.,therapeutically and/or prophylactically active agents). The crystallineforms of Compound 3 or compositions can be administered in combinationwith additional pharmaceutical agents that improve their activity (e.g.,activity in preventing and/or treating a disease associated withaberrant signaling of a growth factor (e.g., VEGF) or with abnormalangiogenesis in a subject, in inhibiting aberrant signaling of a growthfactor (e.g., VEGF) in a subject or cell, or in inhibiting abnormalangiogenesis in a subject), bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body of a subject. It will also be appreciated that thetherapy employed may achieve a desired effect for the same disorder,and/or it may achieve different effects.

The crystalline form of Compound 3 or composition of the invention canbe administered concurrently with, prior to, or subsequent to one ormore additional pharmaceutical agents, which may be useful as, e.g.,combination therapies. Pharmaceutical agents include therapeuticallyactive agents. Pharmaceutical agents also include prophylacticallyactive agents. Pharmaceutical agents include small organic moleculessuch as drug compounds (e.g., compounds approved for human or veterinaryuse by the U.S. Food and Drug Administration as provided in the Code ofFederal Regulations (CFR)), peptides, proteins, carbohydrates,monosaccharides, oligosaccharides, polysaccharides, nucleoproteins,mucoproteins, lipoproteins, synthetic polypeptides or proteins, smallmolecules linked to proteins, glycoproteins, steroids, nucleic acids,DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisenseoligonucleotides, lipids, hormones, vitamins, and cells. In certainembodiments, the additional pharmaceutical agent is a pharmaceuticalagent useful for treating and/or preventing a disease described herein.Each additional pharmaceutical agent may be administered at a doseand/or on a time schedule determined for that pharmaceutical agent. Theadditional pharmaceutical agents may also be administered together witheach other and/or with the compound or composition described herein in asingle dose or administered separately in different doses. Theparticular combination to employ in a regimen will take into accountcompatibility of the inventive compound with the additionalpharmaceutical agent(s) and/or the desired therapeutic and/orprophylactic effect to be achieved. In general, it is expected that theadditional pharmaceutical agent(s) utilized in combination be utilizedat levels that do not exceed the levels at which they are utilizedindividually. In some embodiments, the levels utilized in combinationwill be lower than those utilized individually.

The additional pharmaceutical agents include, but are not limited to,anti-proliferative agents (e.g., anti-cancer agents), anti-angiogenesisagents, anti-inflammatory agents, immunosuppressants, anti-bacterialagents, anti-viral agents, anti-diabetic agents, anti-allergic agents,and pain-relieving agents. In certain embodiments, the additionalpharmaceutical agent is a growth factor inhibitor. In certainembodiments, the additional pharmaceutical agent is a VEGF inhibitor. Incertain embodiments, the additional pharmaceutical agent is anangiogenesis inhibitor. In certain embodiments, the additionalpharmaceutical agent is an endogenous angiogenesis inhibitor (e.g.,vascular endothelial growth factor receptor 1 (VEGFR-1, e.g., pazopanib(VOTRIENT®), cediranib (RECENTIN®), tivozanib (AV-951), axitinib(INLYTA®), semaxanib), HER2 (lapatinib (TYKERB®, TYVERB®), linifanib(ABT-869), MGCD-265, and KRN-633), VEGFR-2 (e.g., regorafenib (BAY73-4506), telatinib (BAY 57-9352), vatalanib (PTK787, PTK/ZK), MGCD-265,OSI-930, and KRN-633), NRP-1, angiopoietin 2, TSP-1, TSP-2, angiostatin,endostatin, vasostatin, calreticulin, platelet factor-4, TIMP, CDAI,Meth-1, Meth-2, IFN-α, IFN-β, IFN-γ, CXCL10, IL-4, IL-12, IL-18,prothrombin (kringle domain-2), antithrombin III fragment, prolactin,VEGI, SPARC, osteopontin, maspin, canstatin, a proliferin-relatedprotein, sorafenib (NEXAVAR®), and restin). In certain embodiments, theadditional pharmaceutical agent is an exogenous angiogenesis inhibitor(e.g., bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101,IFN-α, IL-12, platelet factor-4, suramin, SU5416, thrombospondin, aVEGFR antagonist, an angiostatic steroid, an angiostaticsteroid+heparin, a cartilage-derived angiogenesis inhibitory factor, amatrix metalloproteinase inhibitor, angiostatin, endostatin,2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide,thrombospondin, prolactin, a α_(v)β₃ inhibitor, linomide, andtasquinimod). In certain embodiments, the additional pharmaceuticalagent is a corticosteroid, a receptor tyrosine kinase (RTK) inhibitor, acyclooxygenase (COX) inhibitor, a prostaglandin analog, a non-steroidalanti-inflammatory drug (NSAID), a beta blocker, or a carbonic anhydraseinhibitor. In certain embodiments, the additional pharmaceutical agentis a pharmaceutical agent useful for treating and/or preventing AMD,such as verteporfin (e.g., CHLORIN®, VISUDYNE®), thalidomide (e.g.,AMBIODRY®, SYNOVIR®, THALOMID®), talaporfin sodium (e.g., APTOCINE®,LASERPHYRIN®, LITX®), ranibizumab (e.g., LUCENTIS®), pegaptaniboctasodium (e.g., MACUGEN®, MACUVERSE®), isopropyl unoprostone (e.g.,OCUSEVA®, RESCULA®), interferon beta (e.g., FERON®), fluocinoloneacetonide (e.g., ENVISION TD®, RETISERT®), everolimus (e.g., AFINITOR®,CERTICAN®, VOTUBIA®, ZORTRESS®), eculizumab (e.g., SOLARIS®, SOLIRIS®),dexamethasone (e.g., OSURDEX®, OZURDEX®, POSURDEX®, SURODEX®),canakinumab (e.g., ILARIS®), bromfenac (BROMDAY®), ophthalmic (e.g.,BRONAC®, BRONUCK®, XIBROM®, YELLOX®), brimonidine (e.g., ALPHAGAN®,BROMOXIDINE®, ENIDIN®), anecortave acetate (e.g., RETAANE®, EDEX®,PROSTAVASIN®, RIGIDUR®, VASOPROST®, VIRIDAL®), aflibercept ophthalmicsolution (e.g., EYELEA®, EYLEA®, VEGF-TRAP-EYE®), ocriplasmin (e.g.,ILUVIEN®, MEDIDUR®, MEDIDUR FA®), sirolimus (e.g., PERCEIVA®), NT-501,KH-902, fosbretabulin tromethamine (e.g., ZYBRESTAT®), AL-8309,aganirsen (e.g., NORVESS®), volociximab (e.g., OPTHOTEC®), triamcinolone(e.g., Icon Bioscience), TRC-105, Burixafor (e.g., TG-0054), TB-403(e.g., R-7334), squalamine (e.g., EVIZON®), SB-623, S-646240,RTP-801i-14 (e.g., PF-4523655), RG-7417 (e.g., FCFD-4515S), AL-78898A(e.g., POT-4), PG-11047 (e.g., CGC-11047), pazopanib hydrochloride,sonepcizumab (e.g., ASONEP®, SPHINGOMAB®), padeliporfin (e.g., STAKEL®),OT-551, ontecizumab, NOX-A12, hCNS-SC, Neu-2000, NAFB001, MA09-hRPE,LFG-316, iCo-007 (e.g., ISIS-13650), hl-con1, GSK-933776A, GS-6624(e.g., AB-0024), ESBA-1008, epitalon, E-10030 (e.g., ARC-127),dalantercept, MP-0112, CNTO-2476, CERE-120, AAV-NTN, CCX-168,Brimonidine-DDS, bevasiranib sodium (e.g., Cand5), bertilimumab,AVA-101, ALG-1001, AL-39324, AGN-150998, ACU-4429, A6 (e.g., PARALIT®),TT-30, sFLT-01 gene therapy, RETINOSTAT®, PRS-050 (e.g., ANGIOCAL®),PF-4382923, Palomid-529, MC-1101, GW-824575, Dz13 (e.g., TRC-093), D93,CDX-1135 (e.g., TP10), ATL-1103, ARC-1905, XV-615, wet-AMD antibodies(e.g., pSivida), VEGF/rGel, VAR-10200, VAL-566-620-MULTI, TKI, TK-001,STP-601, dry AMD stem cell therapy (e.g., EyeCyte), OpRegen, SMT-D004,SAR-397769, RTU-007, RST-001, RGNX-004, RFE-007-CAI, retinaldegeneration programme (e.g., ORPHAGEN), retinal cells (e.g., ISCO),ReN003, PRM-167, ProDex, Photoswitches (e.g., Photoswitch Biosciences),Parkinson's therapy, OMS-721, OC-10X, NV. AT.08, NT-503, NAFB002, NADPHoxidase inhibitors (e.g., Alimera Sciences), MC-2002, lyciumanti-angiogenic proteoglycan, IXSVEGF, integrin inhibitors, GW-771806,GBS-007, Eos-013, EC-400, dry-AMD therapy (e.g., Neuron Systems),CGEN-25017, CERE-140, AP-202, AMD therapy (e.g., Valens Therapeutics),AMD therapy (e.g., Amarna Therapeutics), AMD RNAi therapy (e.g., RXi),ALK-001, AMD therapy (e.g., Aciont), AC-301, 4-IPP, zinc-monocysteinecomplexes (e.g., Adeona), vatalanib, TG-100-344, prinomastat, PMX-53,Neovastat, mecamylamine, JSM-6427, JPE-1375, CereCRIB, BA-285, ATX-S10,AG-13958, verteporfin/alphavβ3 conjugate, VEGF/rGel, VEGF-saporin,VEGF-R2 antagonist (e.g., Allostera), VEGF inhibitors (e.g., Santen),VEGF antagonists (e.g., Ark), VANGIOLUX®, Triphenylmethanes (e.g.,Alimera), TG-100-801, TG-100-572, TA-106, T2-TrpRS, SU-0879, stem celltherapy (e.g., Pfizer and UCL), SOD mimetics (e.g., Inotek), SHEF-1,rostaporfin (e.g., PHOTREX®, PURLYTIN®, SnET2), RNA interference (e.g.,Idera and Merck), rhCFHp (e.g., Optherion), retino-NPY, retinitispigmentosa therapy (e.g., Mimetogen), AMD gene therapy (e.g., Novartis),retinal gene therapy (e.g., Genzyme), AMD gene therapy (e.g.,Copernicus), retinal dystrophy ther (e.g., Fovea and Genzyme), Ramotproject No. K-734B, PRS-055, porcine RPE cells (e.g., GenVec), PMI-002,PLG-101 (e.g., BiCentis®), PJ-34, PI3K conjugates (e.g., Semafore),PhotoPoint, Pharmaprojects No. 6526, pegaptanib sodium (e.g.,SurModics®), PEDF ZFP TF, PEDF gene therapy (e.g., GenVec), PDS-1.0,PAN-90806, Opt-21, OPK-HVB-010, OPK-HVB-004, Ophthalmologicals (e.g.,Cell NetwoRx), ophthalmic compounds (e.g., AstraZeneca and Alcon),OcuXan, NTC-200, NT-502, NOVA-21012, NEUROSOLVE®, neuroprotective (e.g.,BDSI), MEDI-548, MCT-355, MCEYE®, LENTIVUE®, LYN-002, LX-213, lutetiumtexaphyrin (e.g., ANTRIN®), LG-339 inhibitors (e.g., Lexicon), KDRkinase inhibitors (e.g., Merck), ISV-616, INDUS-815C, ICAM-1 aptamer(e.g., Eyetech), hedgehog antagonists (e.g., Opthalmo), GTx-822, GS-102,Granzyme B/VEGF®, gene therapy (e.g., EyeGate), GCS-100 analogueprogramme, FOV-RD-27, fibroblast growth factor (e.g., Ramot),fenretinide, F-200 (e.g., Eos-200-F), PANZEM SR®, ETX-6991, ETX-6201,EG-3306, Dz-13, disulfiram (e.g., ORA-102), Diclofenac (e.g.,Ophthalmopharma), ACU-02, CLT-010, CLT-009, CLT-008, CLT-007, CLT-006,CLT-005, CLT-004, CLT-003 (e.g., CHIROVIS®), CLT-001, CETHRIN® (e.g.,BA-210), celecoxib, CD91 antagonist (e.g., Ophthalmophar), CB-42, BNC-4,bestrophin, batimastat, BA-1049, AVT-2, AVT-1, atu012, Ape1 programme(e.g., ApeX-2), anti-VEGF (e.g., Gryphon), AMD ZFPs (e.g., ToolGen), AMDtherapy (e.g., Optherion), AMD therapy (e.g., ItherX), dry AMD therapy(e.g., Opko), AMD therapy (e.g., CSL), AMD therapies (e.g., Pharmacopeiaand Allergan), AMD therapeutic protein (e.g., ItherX), AMD RNAi therapy(e.g., BioMolecular Therapeutics), AM-1101, ALN-VEG01, AK-1003,AGN-211745, ACU-XSP-001 (e.g., EXCELLAIR®), ACU-HTR-028, ACU-HHY-011,ACT-MD (e.g., NewNeural), ABCA4 modulators (e.g., Active Pass), A36(e.g., Angstrom), 267268 (e.g., SB-267268), bevacizumab (e.g., VASTIN®),aflibercept (e.g., EYLEA®), 131-I-TM-601, vandetanib (e.g., CAPRELSA®,ZACTIMA®, ZICTIFA®), sunitinib malate (e.g., SUTENE®, SUTENT®),sorafenib (e.g., NEXAVAR®), pazopanib (e.g., ARMALA®, PATORMA®,VOTRIENT®), axitinib (e.g., INLYTA®), tivozanib, XL-647, RAF-265,pegdinetanib (e.g., ANGIOCEPT®), pazopanib, MGCD-265, icrucumab,foretinib, ENMD-2076, BMS-690514, regorafenib, ramucirumab, plitidepsin(e.g., APLIDIN®), orantinib, nintedanib (e.g., VARGATEF®), motesanib,midostaurin, linifanib, telatinib, lenvatinib, elpamotide, dovitinib,cediranib (e.g., RECENTIN®), JI-101, cabozantinib, brivanib, apatinib,ANGIOZYME®, X-82, SSR-106462, rebastinib, PF-337210, IMC-3C5, CYC116,AL-3818, VEGFR-2 inhibitor (e.g., AB Science), VEGF/rGel (e.g., ClaytonBiotechnologies), TLK-60596, TLK-60404, R84 antibody (e.g., Peregrine),MG-516, FLT4 kinase inhibitors (e.g., Sareum), flt-4 kinase inhibitors,Sareum, DCC-2618, CH-330331, XL-999, XL-820, vatalanib, SU-14813,semaxanib, KRN-633, CEP-7055, CEP-5214, ZK-CDK, ZK-261991, YM-359445,YM-231146, VEGFR-2 kinase inhibitors (e.g., Takeda), VEGFR-2 kinaseinhibitors (e.g., Hanmi), VEGFR-2 antagonist (e.g., Affymax), VEGF/rGel(e.g., Targa), VEGF-TK inhibitors (e.g., AstraZeneca), tyrosine kinaseinhibitors (e.g., Abbott), tyrosine kinase inhibitors (e.g., Abbott),Tie-2 kinase inhibitors (e.g., GSK), SU-0879, SP-5.2, sorafenib bead(e.g., NEXAVAR® bead), SAR-131675, Ro-4383596, R-1530, PharmaprojectsNo. 6059, OSI-930, OSI-817, OSI-632, MED-A300, L-000021649, KM-2550,kinase inhibitors (e.g., MethylGene), kinase inhibitors (e.g., Amgen),Ki-8751, KDR kinase inhibitors (e.g., Celltech), KDR kinase inhibitors(e.g., Merck), KDR kinase inhibitors (e.g., Amgen), KDR inhibitors(e.g., Abbott), KDR inhibitor (e.g., LGLS), JNJ-17029259, IMC-1C11, Flt3/4 anticancer (e.g., Sentinel), EG-3306, DP-2514, DCC-2157, CDP-791,CB-173, c-kit inhibitors (e.g., Deciphera), BIW-8556, anticancers (e.g.,Bracco and Dyax), anti-Flt-1 MAbs (e.g., ImClone), AGN-211745, AEE-788,or AB-434. In certain embodiments, the additional pharmaceutical agentis a pharmaceutical agent useful for treating and/or preventing dry eye,such as cyclosporine (RESTASIS®). In certain embodiments, the additionalpharmaceutical agent is a pharmaceutical agent useful for treatingand/or preventing cystoid macular edema (CME), such as an NSAID (e.g.,bromfenac (BROMDAY®)). In certain embodiments, the additionalpharmaceutical agent is a pharmaceutical agent useful for treatingand/or preventing diabetic macular edema (DME), such as ranibizumab(LUCENTIS®). In certain embodiments, the additional pharmaceutical agentis a pharmaceutical agent useful for treating and/or preventing uveitis,such as TOBRADEX® (0.1% dexamethasone/0.3% tobramycin), ZYLET® (0.5%loteprednol etabonate/0.3% tobramycin)), triamcinolone acetonide(TRIVARIS® and TRIESENCE®), fluocinolone acetonide (RETISERTt®), anddexamethasone (OZURDEX®). In certain embodiments, the additionalpharmaceutical agent is a pharmaceutical agent useful for treatingand/or preventing glaucoma, such as latanoprost (XALATAN®), bimatoprost(LUMIGAN®), travoprost (TRAVATAN Z®), timolol (TIMOPTIC®), brimonidinetartrate (ALPHAGAN®), dorzolamide (TRUSOPT®), and pilocarpine (ISOPTO®).In certain embodiments, the additional pharmaceutical agent is apharmaceutical agent useful for treating and/or preventing an ocularinflammatory disease (e.g., post-surgical inflammation), such assteroids (e.g., loteprednol etabonate (LOTEMAX®), difluprednate(DUREZOL®), prednisolone acetate (PRED MILD® and OMNIPRED®) and NSAIDs(e.g., bromfenac (BROMDAY®), nepafenac (NEVANAC®), ketorolactromethamine (ACULAR LS®, ACUVAIL®, TORADOL®, and SPRIX®), diclofenac(VOLTRAN®, ACLONAC®, and CATAFLAM®).

Also encompassed by the invention are kits (e.g., pharmaceutical packs).The kits provided may comprise a pharmaceutical composition orcrystalline form of Compound 3 of the invention and a container (e.g., avial, ampule, bottle, syringe, and/or dispenser package, or othersuitable container). In some embodiments, provided kits may optionallyfurther include a second container comprising a pharmaceutical excipientfor dilution or suspension of a pharmaceutical composition orcrystalline form of Compound 3. In some embodiments, a pharmaceuticalcomposition or crystalline form of Compound 3 provided in the firstcontainer and the second container are combined to form one unit dosageform.

Thus, in one aspect, provided are kits including a first containercomprising a crystalline form of Compound 3 described herein, or apharmaceutical composition thereof. In certain embodiments, the kitsdescribed herein are useful for preventing and/or treating a diseasedescribed herein. In certain embodiments, the kits described herein areuseful for preventing and/or treating a disease associated with aberrantsignaling of a growth factor (e.g., VEGF) in a subject in need thereof.In certain embodiments, the kits described herein are useful forpreventing and/or treating a disease associated with abnormalangiogenesis in a subject in need thereof. In certain embodiments, thekits described herein are useful for preventing and/or treatingproliferative diseases (e.g., cancers, benign neoplasms, inflammatorydiseases, autoimmune diseases) and/or ocular diseases (e.g., maculardegeneration, glaucoma, diabetic retinopathy, retinoblastoma, edema,uveitis, dry eye, or post-surgical inflammation). In certainembodiments, the kits described herein are useful for inhibitingaberrant signaling of a growth factor (e.g., VEGF) in a subject or cellin need thereof. In certain embodiments, the kits described herein areuseful for inhibiting abnormal angiogenesis in a subject in needthereof. In certain embodiments, the kits further include instructionsfor administering the crystalline form of Compound 3, or thepharmaceutical composition thereof. The kits may also includeinformation as required by a regulatory agency such as the U.S. Food andDrug Administration (FDA). In certain embodiments, the informationincluded in the kits is prescribing information. In certain embodiments,the kits and instructions provide for treating and/or preventing adisease described herein. In certain embodiments, the kits andinstructions provide for preventing and/or treating a disease associatedwith aberrant signaling of a growth factor (e.g., VEGF) in a subject inneed thereof. In certain embodiments, the kits and instructions providefor preventing and/or treating a disease associated with abnormalangiogenesis in a subject in need thereof. In certain embodiments, thekits and instructions provide for inhibiting aberrant signaling of agrowth factor (e.g., VEGF) in a subject or cell in need thereof. Incertain embodiments, the kits and instructions provide for inhibitingabnormal angiogenesis in a subject in need thereof. The kit of theinvention may include one or more additional pharmaceutical agentsdescribed herein as a separate composition.

Also provided by the present invention are particles comprising acrystalline form of Compound 3 described herein that may penetratemucus, pharmaceutical compositions thereof, kits, and methods of usingand preparing the particles, and pharmaceutical compositions thereof.The pharmaceutical compositions, kits, and methods may involve modifyingthe surface coatings of particles, such as particles of pharmaceuticalagents that have a low aqueous solubility. Such pharmaceuticalcompositions, kits, and methods can be used to achieve efficienttransport of particles comprising the inventive crystalline forms ofCompound 3 through mucus barriers in a subject.

In certain embodiments, the crystalline forms, particles, pharmaceuticalcompositions, kits, and methods of the invention are useful forapplications in the eye, such as treating and/or preventing an oculardisease (e.g., macular degeneration, retinopathy, macular edema, retinalvein occlusion, dry eye syndrome, uveitis, allergic conjunctivitis,glaucoma, and rosacea).

The particles (e.g., nanoparticles and microparticles) of the inventioncomprise a crystalline form of Compound 3. In one particular aspect, theparticles comprise crystalline Form B of Compound 3. The particles ofthe invention also include a surface-altering agent that modifies thesurface of the particles to reduce the adhesion of the particles tomucus and/or to facilitate penetration of the particles through mucus.

The present invention also provides pharmaceutical compositionscomprising the inventive particles. In certain embodiments, thepharmaceutical compositions of the invention can be topicallyadministered to the eye of a subject. Topical pharmaceuticalcompositions are advantageous over pharmaceutical compositions that areadministered by injection or orally.

Particles

The present invention also provides pharmaceutical compositionscomprising a plurality of particles or crystals of the invention, whichmay be mucus-penetrating particles or crystals (MPPs). MPPs comprisingcrystalline Form A or crystalline Form B of Compound 3 useful in thepresent invention can be made as described, for example, in U.S. patentPublication Nos. 2013/0316001, 2013/0316006, 2013/0323179, 2013/0316009,2012/0121718, 2010/0215580, and 2008/0166414, which are hereinincorporated by reference in their entirety. Such pharmaceuticalscompositions may be suitable for administration by various routesdescribed herein. In one embodiment, pharmaceutical compositionscomprising a plurality of particles comprising crystalline Form A orcrystalline Form B of Compound 3, wherein the particles aremucus-penetrating particles, are formulated for delivery to the eye of asubject or to treat and/or prevent an ocular disease of a subject. In apreferred embodiment, the mucus-penetrating particles comprisecrystalline Form B of Compound 3.

In some embodiments, the particles of the invention have a core-shelltype configuration. The core may comprise a crystalline form of Compound3, a polymeric carrier, a lipid, and/or a protein. The core may alsocomprise a gel or a liquid.

In some embodiments, the core is a solid. The solid may be, for example,a crystalline form of Compound 3 (e.g., crystalline Form B). In certainembodiments, the core is a gel or liquid (e.g., an oil-in-water orwater-in-oil emulsion).

A crystalline form of Compound 3 (e.g., crystalline Form B) may bepresent in the core in any suitable amount, e.g., at least about 0.01 wt%, at least about 0.1 wt %, at least about 1 wt %, at least about 5 wt%, at least about 10 wt %, at least about 20 wt %, at least about 30 wt%, at least about 40 wt %, at least about 50 wt %, at least about 60 wt%, at least about 70 wt %, at least about 80 wt %, at least about 85 wt%, at least about 90 wt %, at least about 95 wt %, or at least about 99wt % of the core. In one embodiment, the core is formed of 100 wt % of acrystalline form of Compound 3. In some cases, crystalline form ofCompound 3 (e.g., crystalline Form B) may be present in the core at lessthan or equal to about 100 wt %, less than or equal to about 95 wt %,less than or equal to about 90 wt %, less than or equal to about 85 wt%, less than or equal to about 80 wt %, less than or equal to about 70wt %, less than or equal to about 60 wt %, less than or equal to about50 wt %, less than or equal to about 40 wt %, less than or equal toabout 30 wt %, less than or equal to about 20 wt %, less than or equalto about 10 wt %, less than or equal to about 5 wt %, less than or equalto about 2 wt %, or less than or equal to about 1 wt % of the core.Combinations of the above-referenced ranges are also possible (e.g.,present in an amount of at least about 80 wt % and less than or equal toabout 100 wt % of the core). Other ranges are also possible. In oneembodiment, a crystalline form of Compound 3 (e.g., crystalline Form B)comprises at least 90 wt % of the core of a particle of the invention.In another embodiment, a crystalline form of Compound 3 (e.g.,crystalline Form B) comprises at least 95 wt % of the core of a particleof the invention.

When a polymer is present in the core, the polymer may be present in thecore in any suitable amount, e.g., less than about 100 wt %, less thanabout 80 wt %, less than about 60 wt %, less than about 50 wt %, lessthan about 40 wt %, less than about 30 wt %, less than about 20 wt %,less than about 10 wt %, less than about 5 wt %, or less than about 1 wt%. In some cases, the polymer may be present in an amount of at leastabout 1 wt %, at least about 5 wt %, at least about 10 wt %, at leastabout 20 wt %, at least about 30 wt %, at least about 40 wt %, at leastabout 50 wt %, at least about 75 wt %, at least about 90 wt %, or atleast about 99 wt % in the core. Combinations of the above-referencedranges are also possible (e.g., present in an amount of at least about 1wt % and less than about 20 wt %). Other ranges are also possible. Insome embodiments, the core is substantially free of a polymericcomponent.

The core may have any suitable shape and/or size. For instance, the coremay be substantially spherical, non-spherical, oval, rod-shaped,pyramidal, cube-like, disk-shaped, wire-like, or irregularly shaped. Thecore may have a largest or smallest cross-sectional dimension of, forexample, less than about 10 μm, less than about 3 μm, less than about 1μm, less than about 500 nm, less than 400 nm, less than 300 nm, lessthan about 200 nm, less than about 100 nm, less than about 30 nm, orless than about 10 nm. In some cases, the core may have a largest orsmallest cross-sectional dimension of, for example, at least about 10nm, at least about 30 nm, at least about 100 nm, at least about 200 nm,at least about 300 nm, at least about 400 nm, at least about 500 nm, atleast about 1 μm, or at least about 3 μm. Combinations of theabove-referenced ranges are also possible (e.g., a largest or smallestcross-sectional dimension of at least about 30 nm and less than about500 nm). Other ranges are also possible. In some embodiments, the sizesof the cores formed by a process described herein have a Gaussian-typedistribution. Unless indicated otherwise, the measurements of theparticle sizes or core sizes refer to the smallest cross-sectionaldimension.

Techniques to determine sizes (e.g., smallest or largest cross-sectionaldimensions) of particles are known in the art. Examples of suitabletechniques include dynamic light scattering (DLS), transmission electronmicroscopy, scanning electron microscopy, electroresistance counting andlaser diffraction. Although many methods for determining sizes ofparticles are known, the sizes described herein (e.g., average particlesizes and thicknesses) refer to ones measured by DLS.

In some embodiments, a substantial portion of the core is formed of acrystalline form of Compound 3 as described herein that can lead tocertain beneficial and/or therapeutic effects. The core may be, forexample, a nanocrystal (i.e., a nanocrystalline particle) of acrystalline form of Compound 3. In certain embodiments, the coreincludes a polymeric carrier, optionally with a crystalline form ofCompound 3 encapsulated or otherwise associated with the core. Incertain embodiments, the core includes a lipid, protein, gel, liquid,and/or another suitable material to be delivered to a subject. The coreincludes a surface to which one or more surface-altering agents can beattached.

In some embodiments, the core is surrounded by coating, which includesan inner surface and an outer surface. The coating may be formed, atleast in part, of one or more surface-altering agents, such as a polymer(e.g., a block copolymer and/or a polymer having pendant hydroxylgroups), which may associate with the surface of the core. Thesurface-altering agent may be associated with the core particle by, forexample, being covalently attached to the core particle, non-covalentlyattached to the core particle, adsorbed to the core, or attached to thecore through ionic interactions, hydrophobic and/or hydrophilicinteractions, electrostatic interactions, van der Waals interactions, orcombinations thereof.

The coating and/or surface-altering agent of the particles of theinvention may comprise any suitable material, such as a hydrophobicmaterial, a hydrophilic material, and/or an amphiphilic material. Insome embodiments, the coating includes a polymer. In certainembodiments, the polymer is a synthetic polymer (i.e., a polymer notproduced in nature). In other embodiments, the polymer is a naturalpolymer (e.g., a protein, polysaccharide, or rubber). In certainembodiments, the polymer is a surface active polymer. In certainembodiments, the polymer is a non-ionic polymer. In certain embodiments,the polymer is a linear synthetic non-ionic polymer. In certainembodiments, the polymer is a non-ionic block copolymer. The polymer maybe a copolymer. In certain embodiments, one repeat unit of the copolymeris relatively hydrophobic and another repeat unit of the copolymer isrelatively hydrophilic. The copolymer may be, for example, a diblock,triblock, alternating, or random copolymer. The polymer may be chargedor uncharged.

Non-limiting examples of suitable polymers of the coating may includepolyamines, polyethers, polyamides, polyesters, polycarbamates,polyureas, polycarbonates, polystyrenes, polyimides, polysulfones,polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines,polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles,and polyarylates. Non-limiting examples of specific polymers includepoly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA),poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid)(PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lacticacid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA),poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),poly(ethylene glycol), poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene terephthalates such as poly(ethylene terephthalate),polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such aspoly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride)(PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS),polyurethanes, derivatized celluloses such as alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers ofacrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) (jointlyreferred to herein as “polyacrylic acids”), and copolymers and mixturesthereof, polydioxanone and its copolymers, polyhydroxyalkanoates,polypropylene fumarate), polyoxymethylene, poloxamers,poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate.

The molecular weight of the polymer of the coating may vary. In someembodiments, the molecular weight of the polymer of the coating is atleast about 0.5 kDa, at least about 1 kDa, at least about 1.8 kDa, atleast about 2 kDa, at least about 3 kDa, at least about 4 kDa, at leastabout 5 kDa, at least about 6 kDa, at least about 8 kDa, at least about10 kDa, at least about 12 kDa, at least about 15 kDa, at least about 20kDa, at least about 30 kDa, at least about 40 kDa, or at least about 50kDa. In some embodiments, the molecular weight of the polymer of thecoating is less than about 50 kDa, less than about 40 kDa, less thanabout 30 kDa, less than about 20 kDa, less than about 12 kDa, less thanabout 10 kDa, less than about 8 kDa, less than about 6 kDa, less thanabout 5 kDa, or less than about 4 kDa. Combinations of theabove-referenced ranges are possible (e.g., a molecular weight of atleast about 2 kDa and less than about 15 kDa). Other ranges are alsopossible. The molecular weight of the polymer of the coating may bedetermined using any known technique such as light-scattering and gelpermeation chromatography. Other methods are known in the art. Althoughthe particles of the invention, and the coating thereof, may eachinclude polymers, in some embodiments, the particles of the inventioncomprise a hydrophobic material that is not a polymer or pharmaceuticalagent. Non-limiting examples of non-polymeric hydrophobic materialsinclude, for example, metals, waxes, and organic materials (e.g.,organic silanes and perfluorinated or fluorinated organic materials).

In some embodiments, the surface-altering agents, or portions thereof,are chosen to facilitate transport of the particle through or into amucosal barrier (e.g., mucus or a mucosal membrane). In certainembodiments described herein, one or more surface-altering agents areoriented in a particular configuration in the coating. In someembodiments, in which a surface-altering agent is a triblock copolymer,such as a triblock copolymer having a (hydrophilic block)-(hydrophobicblock)-(hydrophilic block) configuration, a hydrophobic block may beoriented towards the surface of the core, and hydrophilic blocks may beoriented away from the core surface (e.g., towards the exterior of theparticle). The hydrophilic blocks may have characteristics thatfacilitate transport of the particle through a mucosal barrier. Theparticular chemical makeup and/or components of the coating andsurface-altering agent(s) can be chosen so as to impart certainfunctionality to the particles, such as enhanced transport throughmucosal barriers.

In some embodiments, at least one particle of the invention includes acore and a coating surrounding the core. A particle including a core anda coating on the core is referred to as a “coated particle.” In certainembodiments, at least one particle of the invention includes a core butnot a coating on the core. A particle including a core but not a coatingon the core is referred to as an “uncoated particle.”

It should be understood that a coating which surrounds a core need notcompletely surround the core, although such embodiments may be possible.For example, the coating may surround at least about 10%, at least about30%, at least about 50%, at least about 70%, at least about 90%, or atleast about 99% of the surface area of a core. In some cases, thecoating substantially surrounds a core. In other cases, the coatingcompletely surrounds a core. In other embodiments, a coating surroundsless than about 100%, less than about 90%, less than about 70%, or lessthan about 50% of the surface area of a core. Combinations of theabove-referenced ranges are also possible (e.g., surrounding at least70% and less than 100% of the surface area of a core).

The material of the coating may be distributed evenly across a surfaceof the core in some cases, and unevenly in other cases. For example, thecoating may include portions (e.g., holes) that do not include anymaterial. If desired, the coating may be designed to allow penetrationand/or transport of certain molecules and components into or out of thecoating, but may prevent penetration and/or transport of other moleculesand components into or out of the coating. The ability of certainmolecules to penetrate and/or be transported into and/or across acoating may depend on, for example, the packing density of thesurface-altering agents forming the coating and the chemical andphysical properties of the components forming the coating. As describedherein, the coating may include one layer of material (i.e., amonolayer) or multilayers of materials. A single type or multiple typesof surface-altering agent may be present.

The coating of particles of the invention can have any suitablethickness. For example, the coating may have an average thickness of atleast about 1 nm, at least about 3 nm, at least about 10 nm, at leastabout 30 nm, at least about 100 nm, at least about 300 nm, at leastabout 1 μm, or at least about 3 μm. In some cases, the average thicknessof the coating is less than about 3 μm, less than about 1 μm, less thanabout 300 nm, less than about 100 nm, less than about 30 nm, less thanabout 10 nm, or less than about 3 nm. Combinations of theabove-referenced ranges are also possible (e.g., an average thickness ofat least about 1 nm and less than about 100 nm). Other ranges are alsopossible. For particles having multiple coatings, each coating may haveone of the thicknesses described herein.

The pharmaceutical compositions of the invention may allow for thecoating of the particles of the invention with hydrophilicsurface-altering moieties without requiring covalent association of thesurface-altering moieties to the surface of the core. In someembodiments, the core having a hydrophobic surface is coated with apolymer described herein, thereby causing a plurality ofsurface-altering moieties to be on the surface of the core withoutsubstantially altering the characteristics of the core itself. Forexample, the surface altering agent may be present on (e.g., adsorbedto) the outer surface of the core. In other embodiments, asurface-altering agent is covalently linked to the core.

In certain embodiments in which the surface-altering agent is adsorbedonto a surface of the core, the surface-altering agent may be inequilibrium with other molecules of the surface-altering agent insolution, optionally with other components (e.g., in a pharmaceuticalcomposition). In some cases, the adsorbed surface-altering agent may bepresent on the surface of the core at a density described herein. Thedensity may be an average density as the surface altering agent is inequilibrium with other components in solution.

In some embodiments, the present invention relates to coated particlescomprising a core comprising crystalline Form A or crystalline Form B ofCompound 3 described herein and a coating surrounding the core. In someembodiments, the coating comprises a hydrophilic material. The coatingmay comprise one or more surface-altering agents described herein, suchas a polymer and/or a surfactant (e.g., a PVA, a poloxamer, apolysorbate (e.g., TWEEN 80®)). Other coatings or surface-alteringagents useful in the present invention are described, for example, inU.S. patent Publication Nos. 2013/0316001, 2013/0316006, 2013/0323179,2013/0316009, 2012/0121718, 2010/0215580, and 2008/0166414, which areherein incorporated by reference in their entirety.

In some embodiments, the compositions and methods involve the use ofpoloxamers that aid particle transport in mucus. Poloxamers aretypically nonionic triblock copolymers comprising a central hydrophobicblock (e.g., a poly(propylene oxide) block) flanked by two hydrophilicblocks (e.g., poly(ethylene oxide) blocks). Poloxamers have the tradename PLURONIC®. Examples of PLURONIC® polymers that may be useful in theembodiments described herein include, but are not limited to, F127(poloxamer 407), F38, F108 (poloxamer 338), F68, F77, F87, F88, F98,L101, L121, L31, L35, L43, L44, L61, L62, L64, L81, L92, N3, P103, P104,P105, P123, P65, P84, and P85. In certain embodiments, the molecularweight of the hydrophobic block of the triblock copolymer of the(hydrophilic block)-(hydrophobic block)-(hydrophilic block)configuration is at least about 2 kDa, and the two hydrophilic blocksconstitute at least about 15 wt % of the triblock copolymer.

In certain embodiments, the compositions and methods involve the use ofpolysorbates that aid particle transport in mucus. Polysorbates aretypically derived from PEGylated sorbitan (a derivative of sorbitol)esterified with fatty acids. Common brand names for polysorbates includeTWEEN®, ALKEST®, CANARCEL®. Examples of polysorbates includepolyoxyethylene sorbitan monooleate (e.g., TWEEN 80®), polyoxyethylenesorbitan monostearate (e.g., TWEEN 60®), polyoxyethylene sorbitanmonopalmitate (e.g., TWEEN 40®), and polyoxyethylene sorbitanmonolaurate (e.g., TWEEN 20®).

It should be understood that components and configurations other thanthose described herein may be suitable for certain particles andpharmaceutical compositions, and that not all of the componentsdescribed are necessarily present in some embodiments.

In some embodiments, particles of the invention, when introduced into asubject, may interact with one or more components in the subject such asmucus, cells, tissues, organs, particles, fluids (e.g., blood),microorganisms, and portions or combinations thereof. In someembodiments, the coating of the inventive particle can be designed toinclude surface-altering agents or other components with properties thatallow favorable interactions (e.g., transport, binding, and adsorption)with one or more materials from the subject. For example, the coatingmay include surface-altering agents or other components having a certainhydrophilicity, hydrophobicity, surface charge, functional group,specificity for binding, and/or density to facilitate or reduceparticular interactions in the subject. One example is choosing ahydrophilicity, hydrophobicity, surface charge, functional group,specificity for binding, and/or density of one or more surface-alteringagents to reduce the physical and/or chemical interactions between theparticle and mucus of the subject, so as to enhance the mobility of theparticle through mucus. Other examples are described in more detailbelow.

In some embodiments, once a particle is successfully transported intoand/or across a mucosal barrier (e.g., mucus or a mucosal membrane) in asubject, further interactions between the particle and the subject maytake place. In some embodiments, in which the core comprises apharmaceutical agent or compound of the invention, the conversion,breakdown, release, and/or transport of the pharmaceutical agent fromthe particle can lead to certain beneficial and/or therapeutic effectsin the subject. Therefore, the particles of the invention can be usedfor the treatment and/or prevention of certain diseases.

Examples for the use of the particles of the invention are providedbelow in the context of being suitable for administration to a mucosalbarrier (e.g., mucus or a mucosal membrane) in a subject. It should beappreciated that while many of the embodiments herein are described inthis context, and in the context of providing a benefit for diseasesthat involve transport of materials across a mucosal barrier, theinvention is not limited as such, and the particles, pharmaceuticalcompositions, and kits of the invention may be used to treat and/orprevent other diseases.

In some embodiments, the pharmaceutical compositions of the inventioncomprise MPPs that include a crystalline form of Compound 3 andoptionally at least one additional pharmaceutical agent, each of whichis associated with polymer carriers via encapsulation or otherprocesses. In other embodiments, the pharmaceutical compositions of theinvention comprise MPPs without any polymeric carriers or with minimaluse of polymeric carriers. Polymer-based MPPs may have one or moreinherent limitations in some embodiments. In particular, in light ofdrug delivery applications, these limitations may include one or more ofthe following. A) Low drug encapsulation efficiency and low drugloading: encapsulation of drugs into polymeric particles is ofteninefficient, as generally less than 10% of the total amount of drug usedgets encapsulated into particles during manufacturing; additionally,drug loadings above 50% are rarely achieved. B) Convenience of usage:pharmaceutical compositions based on drug-loaded polymeric particles, ingeneral, typically need to be stored as dry powder to avoid prematuredrug release and thus require either point-of-use re-constitution or asophisticated dosing device. C) Biocompatibility: accumulation of slowlydegrading polymer carriers following repeated dosing and their toxicityover the long term present a major concern for polymeric drug carriers.D) Chemical and physical stability: polymer degradation may compromisestability of encapsulated drugs. In many encapsulation processes, thedrug undergoes a transition from a solution phase to a solid phase,which is not well-controlled in terms of physical form of the emergingsolid phase (i.e., amorphous vs. crystalline vs. crystallinepolymorphs); this is a concern for multiple aspects of pharmaceuticalcomposition performance, including physical and chemical stability andrelease kinetics. E) Manufacturing complexity: manufacturing, especiallyscalability, of drug-loaded polymeric MPPs is a fairly complex processthat may involve multiple steps and a considerable amount of toxicorganic solvents. Therefore, by avoiding or minimizing the need toencapsulate pharmaceutical agents into polymeric carriers, certainlimitations of polymeric MPPs with respect to drug loading, convenienceof usage, biocompatibility, stability, and/or complexity ofmanufacturing, may be addressed.

It should be appreciated, however, that in other embodiments,pharmaceutical agents may be associated with polymer carriers viaencapsulation or other processes. Thus, the description provided hereinis not limited in this respect. For instance, despite theabove-mentioned drawbacks of certain mucus-penetrating particlesincluding a polymeric carrier, in certain embodiments such particles maybe preferred. For example, it may be preferable to use polymer carriersfor controlled release purposes and/or for encapsulating certainpharmaceutical agents that are difficult to formulate into particles. Assuch, in some embodiments described herein, particles that include apolymer carrier are described.

In some embodiments, the pharmaceutical compositions of the inventioninvolve the use of poly(vinyl alcohols) (PVAs), a water-solublenon-ionic synthetic polymer, to aid particle transport in mucus, such asdescribed in U.S. patent Publication No. 2013/0316009, which is hereinincorporated by referenced in its entirety. The pharmaceuticalcompositions may involve making MPPs or MPCs by, for example, anemulsification process in the presence of specific PVAs. In certainembodiments, the pharmaceutical compositions and methods involve makingMPPs or MPCs from pre-fabricated particles by non-covalent coating withspecific PVAs. In some embodiments, the pharmaceutical compositions andmethods involve making MPPs in the presence of specific PVAs without anypolymeric carriers or with minimal use of polymeric carriers. It shouldbe appreciated, however, that in other embodiments, polymeric carrierscan be used.

Particles with Reduced Mucoadhesion

Particles of the invention comprising crystalline forms of Compound 3(e.g., crystalline Form B) may have reduced mucoadhesiveness. A materialin need of increased diffusivity through mucus may be hydrophobic, mayinclude many hydrogen bond donors or acceptors, and/or may be highlycharged. In some cases, the material may include a crystalline oramorphous solid material. The material, which may serve as a core, maybe coated with a suitable polymer described herein, thereby forming aparticle with a plurality of surface-altering moieties on the surface,resulting in reduced mucoadhesion. Particles of the invention havingreduced mucoadhesion may alternatively be characterized as havingincreased transport through mucus, being mobile in mucus, ormucus-penetrating (i.e., mucus-penetrating particles), meaning that theparticles are transported through mucus faster than a negative controlparticle. The negative control particle may be a particle that is knownto be mucoadhesive, e.g., an unmodified particle or core that is notcoated with a coating described herein, such as a 200 nm carboxylatedpolystyrene particle.

Particles of the invention may be adapted for delivery (e.g., oculardelivery) to mucus or a mucosal surface of a subject. The particles withsurface-altering moieties may be delivered to the mucosal surface of asubject, may pass through the mucosal barrier in the subject, and/orprolonged retention and/or increased uniform distribution of theparticles at mucosal surfaces, e.g., due to reduced mucoadhesion.

Furthermore, in some embodiments, the particles of the invention havingreduced mucoadhesion facilitate better distribution of the particles atthe surface of a tissue of a subject and/or have a prolonged presence atthe surface of the tissue, compared to particles that are moremucoadhesive. For example, a luminal space such as the gastrointestinaltract is surrounded by a mucus-coated surface. Mucoadhesive particlesdelivered to such a space are typically removed from the luminal spaceand from the mucus-coated surface by the subject's natural clearancemechanisms. The particles of the invention with reduced mucoadhesion mayremain in the luminal space for relatively longer periods compared tothe mucoadhesive particles. This prolonged presence may prevent orreduce clearance of the particles and/or may allow for betterdistribution of the particles on the surface of the tissue. Theprolonged presence may also affect the particle transport through theluminal space, e.g., the particles may distribute into the mucus layerand may reach the underlying epithelium.

In certain embodiments, the core of the particles of the inventioncoated with the polymer of the coating may pass through mucus or amucosal barrier in a subject, exhibit prolonged retention, and/orincrease uniform distribution of the particles at mucosal surfaces,e.g., such substances are cleared more slowly (e.g., at least about 2times, about 5 times, about 10 times, or even at least about 20 timesmore slowly) from a subject's body as compared to a negative controlparticle of the invention.

The mobility of the particles of the invention in mucus may becharacterized in, e.g., the relative velocity and/or diffusivity of theparticles. In certain embodiments, the particles of the invention havecertain relative velocity, <V_(mean)>_(rel), which is defined asfollows:

$\begin{matrix}{{< V_{mean} >_{rel}} = \frac{< V_{mean} >_{Sample}{- {< V_{mean} >_{Neg{ative}\mspace{14mu}{control}}}}}{< V_{mean} >_{{Positive}\mspace{14mu}{control}}{- {< V_{mean} >_{{Negative}\mspace{14mu}{control}}}}}} & ( {{Equation}\mspace{20mu} 1} )\end{matrix}$wherein:

<V_(mean)> is the ensemble average trajectory-mean velocity;

V_(mean) is the velocity of an individual particle averaged over itstrajectory;

the sample is the particle of interest;

the negative control is a 200 nm carboxylated polystyrene particle; and

the positive control is a 200 nm polystyrene particle densely PEGylatedwith 2-5 kDa PEG.

The relative velocity can be measured by a multiple particle trackingtechnique. For instance, a fluorescent microscope equipped with a CCDcamera can be used to capture 15 s movies at a temporal resolution of66.7 ms (15 frames/s) under 100× magnification from several areas withineach sample for each type of particles: sample, negative control, andpositive control. The sample, negative control, and positive control maybe fluorescent particles to observe tracking. Alternativelynon-fluorescent particles may be coated with a fluorescent molecule, afluorescently tagged surface agent, or a fluorescently tagged polymer.An advanced image processing software (e.g., Image Pro or MetaMorph) canbe used to measure individual trajectories of multiple particles over atime-scale of at least 3.335 s (50 frames).

In some embodiments, a particle described herein has a relative velocityof greater than or equal to about 0.3, greater than or equal to about0.4, greater than or equal to about 0.5, greater than or equal to about0.6, greater than or equal to about 0.7, greater than or equal to about0.8, greater than or equal to about 0.9, greater than or equal to about1.0, greater than or equal to about 1.1, greater than or equal to about1.2, greater than or equal to about 1.3, greater than or equal to about1.4, greater than or equal to about 1.5, greater than or equal to about1.6, greater than or equal to about 1.7, greater than or equal to about1.8, greater than or equal to about 1.9 or greater than or equal toabout 2.0 in mucus. In some embodiments, a particle described herein hasa relative velocity of less than or equal to about 10.0, less than orequal to about 8.0, less than or equal to about 6.0, less than or equalto about 4.0, less than or equal to about 3.0, less than or equal toabout 2.0, less than or equal to about 1.9, less than or equal to about1.8, less than or equal to about 1.7, less than or equal to about 1.6,less than or equal to about 1.5, less than or equal to about 1.4, lessthan or equal to about 1.3, less than or equal to about 1.2, less thanor equal to about 1.1, less than or equal to about 1.0, less than orequal to about 0.9, less than or equal to about 0.8, or less than orequal to about 1.7 in mucus. Combinations of the above-noted ranges arepossible (e.g., a relative velocity of greater than or equal to about0.5 and less than or equal to about 6.0). Other ranges are alsopossible. The mucus may be, for example, human cervicovaginal mucus.

In certain embodiments, a particle described herein can diffuse throughmucus or a mucosal barrier at a greater rate or diffusivity than acontrol particle or a corresponding particle (e.g., a correspondingparticle that is unmodified and/or is not coated with a coatingdescribed herein). In some cases, a particle described herein may passthrough mucus or a mucosal barrier at a rate of diffusivity that is atleast about 10 times, 20 times, 30 times, 50 times, 100 times, 200times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times, ormore, higher than a control particle or a corresponding particle. Insome cases, a particle described herein may pass through mucus or amucosal barrier at a rate of diffusivity that is less than or equal toabout 10000 times higher, less than or equal to about 5000 times higher,less than or equal to about 2000 times higher, less than or equal toabout 1000 times higher, less than or equal to about 500 times higher,less than or equal to about 200 times higher, less than or equal toabout 100 times higher, less than or equal to about 50 times higher,less than or equal to about 30 times higher, less than or equal to about20 times higher, or less than or equal to about 10 times higher than acontrol particle or a corresponding particle. Combinations of theabove-referenced ranges are also possible (e.g., at least about 10 timesand less than or equal to about 1000 times higher than a controlparticle or a corresponding particle). Other ranges are also possible.

For the purposes of the comparisons described herein, the correspondingparticles may be approximately the same size, shape, and/or density asthe particles of the invention but lack the coating that makes theparticles of the invention mobile in mucus. In some embodiments, themeasurement of the geometric mean square displacement and rate ofdiffusivity of the particles (e.g., the corresponding particles andparticles of the invention) is based on a time scale of about 1 second,about 3 seconds, or about 10 seconds. Methods for determining thegeometric mean square displacement and rate of diffusivity are known inthe art. The particles of the invention may pass through mucus or amucosal barrier with a geometric mean squared displacement that is atleast about 10 times, about 30 times, about 100 times, about 300 times,about 1000 times, about 3000 times, about 10000 times higher thancorresponding particles or negative control particles. In someembodiments, the particles of the invention pass through mucus or amucosal barrier with a geometric mean squared displacement that is lessthan about 10000 times higher, less than about 3000 times higher, lessthan about 1000 times higher, less than about 300 times higher, lessthan about 100 times higher, less than about 30 times higher, or lessthan about 10 times higher than negative control particles orcorresponding particles. Combinations of the above-referenced ranges arealso possible (e.g., at least about 10 times and less than about 1000times higher than negative control particles or correspondingparticles). Other ranges are also possible.

In some embodiments, particles of the invention diffuse through amucosal barrier at a rate approaching the rate or diffusivity at whichthe particles can diffuse through water. In some embodiments, theparticles of the invention pass through a mucosal barrier at a rate ordiffusivity that is less than about 1/100, less than about 1/300, lessthan about 1/1000, less than about 1/3000, less than about 1/10,000 ofthe diffusivity that the particles diffuse through water under similarconditions. In some embodiments, particles of the invention pass througha mucosal barrier at a rate or diffusivity that is greater than or equalto about 1/10,000, greater than or equal to about 1/3000, greater thanor equal to about 1/1000, greater than or equal to about 1/300, orgreater than or equal to about 1/100 of the diffusivity that theparticles diffuse through water under similar conditions. Combinationsof the above-referenced ranges are also possible (e.g., greater than orequal to about 1/3000 and less than 1/300 the diffusivity that theparticles diffuse through water under similar conditions). Other rangesare also possible. The measurement of diffusivity may be based on a timescale of about 1 second, or about 0.5 second, or about 2 seconds, orabout 5 seconds, or about 10 seconds.

In some embodiments, the particles of the invention diffuse throughhuman cervicovaginal mucus at a diffusivity that is less than about1/500 of the diffusivity that the particles diffuse through water. Insome embodiments, the measurement of diffusivity is based on a timescale of about 1 second, or about 0.5 second, or about 2 seconds, orabout 5 seconds, or about 10 seconds.

In certain embodiments, the present invention provides particles thattravel through mucus, such as human cervicovaginal mucus, at certainabsolute diffusivities. For example, the particles of described hereinmay travel at diffusivities of at least about 1×10⁻⁴ μm/s, 2×10⁻⁴ μm/s,5×10⁻⁴ μm/s, 1×10⁻³ μm/s, 2×10⁻³ μm/s, 5×10⁻³ μm/s, 1×10⁻² μm/s, 2×10⁻²μm/s, 4×10⁻² μm/s, 5×10⁻² μm/s, 6×10⁻² μm/s, 8×10⁻² μm/s, 1×10⁻¹ μm/s,2×10⁻¹ μm/s, 5×10⁻¹ μm/s, 1 μm/s, or 2 μm/s. In some cases, theparticles may travel at diffusivities of less than or equal to about 2μm/s, less than or equal to about 1 μm/s, less than or equal to about5×10⁻¹ μm/s, less than or equal to about 2×10⁻¹ μm/s, less than or equalto about 1×10⁻¹ μm/s, less than or equal to about 8×10⁻² μm/s, less thanor equal to about 6×10⁻² μm/s, less than or equal to about 5×10⁻² μm/s,less than or equal to about 4×10⁻² μm/s, less than or equal to about2×10⁻² μm/s, less than or equal to about 1×10⁻² μm/s, less than or equalto about 5×10⁻³ μm/s, less than or equal to about 2×10⁻³ μm/s, less thanor equal to about 1×10⁻³ μm/s, less than or equal to about 5×10⁻⁴ μm/s,less than or equal to about 2×10⁻⁴ μm/s, or less than or equal to about1×10⁻⁴ μm/s. Combinations of the above-referenced ranges are alsopossible (e.g., greater than or equal to about 2×10⁻⁴ μm/s and less thanor equal to about 1×10⁻¹ μm/s). Other ranges are also possible. In somecases, the measurement is based on a time scale of about 1 second, orabout 0.5 second, or about 2 seconds, or about 5 seconds, or about 10seconds.

It should be appreciated that while the mobility (e.g., relativevelocity and diffusivity) of the particles of the invention may bemeasured in human cervicovaginal mucus, the mobility may be measured inother types of mucus as well.

In certain embodiments, a particle described herein comprisessurface-altering moieties at a given density. The surface-alteringmoieties may be the portions of a surface-altering agent that are, forexample, exposed to the solvent containing the particle. As an example,the hydrolyzed units/blocks of PVA may be surface-altering moieties ofthe surface-altering agent PVA. In another example, the PEG segments maybe surface-altering moieties of the surface-altering agent PEG-PPO-PEG.In some cases, the surface-altering moieties and/or surface-alteringagents are present at a density of at least about 0.001 units ormolecules per nm², at least about 0.002, at least about 0.005, at leastabout 0.01, at least about 0.02, at least about 0.05, at least about0.1, at least about 0.2, at least about 0.5, at least about 1, at leastabout 2, at least about 5, at least about 10, at least about 20, atleast about 50, at least about 100 units or molecules per nm², or moreunits or molecules per nm². In some cases, the surface-altering moietiesand/or surface-altering agents are present at a density of less than orequal to about 100 units or molecules per nm², less than or equal toabout 50, less than or equal to about 20, less than or equal to about10, less than or equal to about 5, less than or equal to about 2, lessthan or equal to about 1, less than or equal to about 0.5, less than orequal to about 0.2, less than or equal to about 0.1, less than or equalto about 0.05, less than or equal to about 0.02, or less than or equalto about 0.01 units or molecules per nm². Combinations of theabove-referenced ranges are possible (e.g., a density of at least about0.01 and less than or equal to about 1 units or molecules per nm²).Other ranges are also possible. In some embodiments, the density valuesdescribed above may be an average density as the surface altering agentis in equilibrium with other components in solution.

Those skilled in the art would be aware of methods to estimate theaverage density of surface-altering moieties (see, for example, Budijonoet al., Colloids and Surfaces A: Physicochem. Eng. Aspects 2010, 360,105-110; Joshi et al., Anal. Chim. Acta 1979, 104,153-160). For example,as described herein, the average density of surface-altering moietiescan be determined using HPLC quantitation and DLS analysis. A suspensionof particles for which surface density determination is of interest isfirst sized using DLS: a small volume is diluted to an appropriateconcentration (e.g., about 100 μg/mL), and the z-average diameter istaken as a representative measurement of particle size. The remainingsuspension is then divided into two aliquots. Using HPLC, the firstaliquot is assayed for the total concentration of core material and forthe total concentration of the surface-altering moiety. Again usingHPLC, the second aliquot is assayed for the concentration of free orunbound surface-altering moiety. In order to get only the free orunbound surface-altering moiety from the second aliquot, the particles,and therefore any bound surface-altering moiety, are removed byultracentrifugation. By subtracting the concentration of the unboundsurface-altering moiety from the total concentration of surface-alteringmoiety, the concentration of bound surface-altering moiety can bedetermined. Since the total concentration of core material was alsodetermined from the first aliquot, the mass ratio between the corematerial and the surface-altering moiety can be determined. Using themolecular weight of the surface-altering moiety the number ofsurface-altering moiety to mass of core material can be calculated. Toturn this number into a surface density measurement, the surface areaper mass of core material needs to be calculated. The volume of theparticle is approximated as that of a sphere with the diameter obtainedfrom DLS allowing for the calculation of the surface area per mass ofcore material. In this way the number of surface-altering moieties persurface area can be determined.

In certain embodiments, the particles of the invention comprisesurface-altering moieties and/or agents that affect the zeta-potentialof the particle. The zeta potential of the particle may be, for example,at least about −100 mV, at least about −30 mV, at least about −10 mV, atleast about −3 mV, at least about 3 mV, at least about 10 mV, at leastabout 30 mV, or at least about 100 mV. The zeta potential of theparticle may also be, for example, less than about 100 mV, less thanabout 30 mV, less than about 10 mV, less than about 3 mV, less thanabout −3 mV, less than about −10 mV, less than about −30 mV, or lessthan about −100 mV. Combinations of the above-referenced ranges arepossible (e.g., a zeta-potential of at least about −30 mV and less thanabout 30 mV). Other ranges are also possible.

The coated particles described herein may have any suitable shape and/orsize. In some embodiments, a coated particle has a shape substantiallysimilar to the shape of the core. In some cases, a coated particledescribed herein may be a nanoparticle, i.e., the particle has acharacteristic dimension of less than about 1 micrometer, where thecharacteristic dimension of the particle is the diameter of a perfectsphere having the same volume as the particle. In other embodiments,larger sizes are possible (e.g., about 1-10 microns). A plurality ofparticles, in some embodiments, may also be characterized by an averagesize (e.g., an average largest cross-sectional dimension, or an averagesmallest cross-sectional dimension for the plurality of particles). Aplurality of particles may have an average size of, for example, lessthan or equal to about 10 μm, less than or equal to about 5 μm, lessthan or equal to about 1 μm, less than or equal to about 800 nm, lessthan or equal to about 700 nm, less than or equal to about 500 nm, lessthan or equal to 400 nm, less than or equal to 300 nm, less than orequal to about 200 nm, less than or equal to about 100 nm, less than orequal to about 75 nm, less than or equal to about 50 nm, less than orequal to about 40 nm, less than or equal to about 35 nm, less than orequal to about 30 nm, less than or equal to about 25 nm, less than orequal to about 20 nm, less than or equal to about 15 nm, or less than orequal to about 5 nm. In some cases, a plurality of particles may have anaverage size of, for example, at least about 5 nm, at least about 20 nm,at least about 50 nm, at least about 100 nm, at least about 200 nm, atleast about 300 nm, at least about 400 nm, at least about 500 nm, atleast about 1 μm, at least or at least about 5 μm. Combinations of theabove-referenced ranges are also possible (e.g., an average size of atleast about 50 nm and less than or equal to about 500 nm). Other rangesare also possible. In some embodiments, the sizes of the cores formed bya process described herein have a Gaussian-type distribution.

Pharmaceutical Agents

A particle or pharmaceutical composition of the invention may compriseat least one pharmaceutically acceptable crystalline form of Compound 3.In one embodiment, a particle or pharmaceutical composition comprisescrystalline Form A. In another embodiment, a particle or pharmaceuticalcomposition comprises crystalline Form B. The crystalline form ofCompound 3 may be present in the core and/or one or more coatings of theparticle (e.g., dispersed throughout the core and/or coating). In someembodiments, the crystalline form of Compound 3 may be disposed on thesurface of the particle (e.g., on the outer or inner surface of the oneor more coatings or on the surface of the core). The crystalline form ofCompound 3 may be contained within the particle and/or disposed in aportion of the particle using commonly known techniques (e.g., coating,adsorption, covalent linkage, and encapsulation). In some embodiments,the crystalline form of Compound 3 is present during the formation ofthe core. In other embodiments, the crystalline form of Compound 3 isnot present during the formation of the core. In certain embodiments,the crystalline form of Compound 3 is present during the coating of thecore.

In some embodiments, the crystalline form of Compound 3 contained in aparticle or pharmaceutical composition of the invention has atherapeutic and/or prophylactic effect in a mucosal tissue to betargeted. Non-limiting examples of mucosal tissues include ophthalmic,respiratory (e.g., including nasal, pharyngeal, tracheal, and bronchialmembranes), oral (e.g., including the buccal and esophageal membranesand tonsil surface), gastrointestinal (e.g., including stomach, smallintestine, large intestine, colon, rectum), nasal, and genital (e.g.,including vaginal, cervical and urethral membranes) tissues.

Any suitable number of pharmaceutical agents may be present in aparticle or pharmaceutical composition of the invention. For example, inaddition to a crystalline form of Compound 3, at least 1, at least 2, atleast 3, at least 4, at least 5, or more pharmaceutical agents may bepresent in the particle or pharmaceutical composition of the invention.In certain embodiments, less than 10 pharmaceutical agents are presentin the particle or pharmaceutical composition of the invention.

In certain embodiments, the pharmaceutical agent in the particles orpharmaceutical compositions of the invention is a crystal form ofCompound 3. In one embodiment, the pharmaceutical agent in the particlesor pharmaceutical compositions of the invention is crystalline Form A ofCompound 3. In another embodiment, the pharmaceutical agent in theparticles or pharmaceutical compositions of the invention is crystallineForm B of Compound 3. The pharmaceutical agent described herein (e.g., acrystalline form of Compound 3) may be encapsulated in a polymer, alipid, a protein, or a combination thereof.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticalcompositions comprising at least one particle of the invention.Pharmaceutical compositions described herein and for use in accordancewith the articles and methods described herein may include apharmaceutically acceptable excipient or carrier. A pharmaceuticallyacceptable excipient or pharmaceutically acceptable carrier may includea non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any suitable type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose, and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose, andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; detergents such as TWEEN 80; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator. As would be appreciated by one of skill in this art, theexcipients may be chosen based on the route of administration asdescribed below, the pharmaceutical agent being delivered, time courseof delivery of the agent, etc.

Pharmaceutical compositions containing the particles described hereinmay be administered to a subject via any route known in the art. Theseinclude, but are not limited to, oral, sublingual, nasal, injection(e.g., intravenous, intradermal, subcutaneous, intramuscular), rectal,vaginal, intraarterial, intracisternally, intraperitoneal, intravitreal,periocular, topical (e.g., ocular or dermal, such as by powders, creams,ointments, or drops), buccal, and inhalational administration. In someembodiments, compositions described herein may be administeredparenterally as injections (intravenous, intramuscular, orsubcutaneous), drop infusion preparations, or suppositories. As would beappreciated by one of skill in this art, the route of administration andthe effective dosage to achieve the desired biological effect may bedetermined by the agent being administered, the target organ, thepreparation being administered, time course of administration, diseasebeing treated, intended use, etc.

In certain embodiments, the pharmaceutical compositions are useful forthe delivery of a crystalline form of Compound 3 described hereinthrough or to mucus or a mucosal surface in a subject. Thepharmaceutical compositions may be delivered to the mucosal surface inthe subject and may pass through a mucosal barrier in the subject (e.g.,mucus), and/or may show prolonged retention and/or increased uniformdistribution of the particles of the invention at the mucosal surface,e.g., due to reduced mucoadhesion. In certain embodiments, thepharmaceutical compositions are useful in increasing the bioavailabilityof Compound 3 in the subject. In certain embodiments, the pharmaceuticalcompositions are useful in increasing the concentration of the Compound3 in the subject. In certain embodiments, the pharmaceuticalcompositions are useful in increasing the exposure of Compound 3 in thesubject. Moreover, the pharmaceutical compositions may be useful intreating and/or preventing a disease (e.g., ocular disease) in asubject.

Moreover, the pharmaceutical compositions may be administeredparenterally as injections (intravenous, intramuscular, orsubcutaneous), drop infusion preparations, or suppositories. Forophthalmic applications, the pharmaceutical compositions may beadministered by injection (e.g., intraocular, conjunctival,subconjunctival, intrastromal, intravitreal, or intracameral), or by thelocal or ophthalmic mucous membrane route, the pharmaceuticalcompositions may be administered topically, such as solutions,suspensions (e.g., eye drops), gels, or ointments.

In some embodiments, particles described herein that may be administeredin inhalant or aerosol formulations comprise one or more pharmaceuticalagents, such as adjuvants, diagnostic agents, imaging agents, ortherapeutic agents useful in inhalation therapy. The particle size ofthe particulate medicament should be such as to permit inhalation ofsubstantially all of the medicament into the lungs upon administrationof the aerosol formulation and may be, for example, less than about 20microns, e.g., in the range of about 1 to about 10 microns, e.g., about1 to about 5 microns, although other ranges are also possible. Theparticle size of the medicament may be reduced by conventional means,for example by milling or micronisation. Alternatively, the particulatemedicament can be administered to the lungs via nebulization of asuspension. The final aerosol formulation may contain, for example,between 0.005-90% w/w, between 0.005-50%, between 0.005-10%, betweenabout 0.005-5% w/w, or between 0.01-1.0% w/w, of medicament relative tothe total weight of the formulation. Other ranges are also possible.

It is desirable, but by no means required, that the formulationsdescribed herein contain no components which may provoke the degradationof stratospheric ozone. In particular, in some embodiments, propellantsare selected that do not contain or do not consist essentially ofchlorofluorocarbons such as CCl₃F, CCl₂F₂, and CF₃CCl₃.

The aerosol may comprise propellant. The propellant may optionallycontain an adjuvant having a higher polarity and/or a higher boilingpoint than the propellant. Polar adjuvants which may be used include(e.g., C₂₋₆) aliphatic alcohols and polyols such as ethanol,isopropanol, and propylene glycol, preferably ethanol. In general, onlysmall quantities of polar adjuvants (e.g., 0.05-3.0% w/w) may berequired to improve the stability of the dispersion—the use ofquantities in excess of 5% w/w may tend to dissolve the medicament.Formulations in accordance with the embodiments described herein maycontain less than 1% w/w, e.g., about 0.1% w/w, of polar adjuvant.However, the formulations described herein may be substantially free ofpolar adjuvants, especially ethanol. Suitable volatile adjuvants includesaturated hydrocarbons such as propane, n-butane, isobutane, pentane andisopentane and alkyl ethers such as dimethyl ether. In general, up to50% w/w of the propellant may comprise a volatile adjuvant, for example,up to 30% w/w of a volatile saturated C₁-C₆ hydrocarbon. Optionally, theaerosol formulations according to the invention may further comprise oneor more surfactants. The surfactants can be physiologically acceptableupon administration by inhalation. Within this category are includedsurfactants such as L-α-phosphatidylcholine (PC),1,2-dipalmitoylphosphatidycholine (DPPC), oleic acid, sorbitantrioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylenesorbitan monolaurate, polyoxyethylene sorbitan monooleate, naturallecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether,lauryl polyoxyethylene ether, block copolymers of oxyethylene andoxypropylene, synthetic lecithin, diethylene glycol dioleate,tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glycerylmonooleate, glyceryl monostearate, glyceryl monoricinoleate, cetylalcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridiniumchloride, benzalkonium chloride, olive oil, glyceryl monolaurate, cornoil, cotton seed oil, and sunflower seed oil.

The formulations described herein may be prepared by dispersal of theparticles in the selected propellant and/or co-propellant in anappropriate container, e.g., with the aid of sonication. The particlesmay be suspended in co-propellant and filled into a suitable container.The valve of the container is then sealed into place and the propellantintroduced by pressure filling through the valve in the conventionalmanner. The particles may be thus suspended or dissolved in a liquefiedpropellant, sealed in a container with a metering valve and fitted intoan actuator. Such metered dose inhalers are well known in the art. Themetering valve may meter 10 to 500 μL and preferably 25 to 150 μL. Incertain embodiments, dispersal may be achieved using dry powder inhalers(e.g., spinhaler) for the particles (which remain as dry powders). Inother embodiments, nanospheres, may be suspended in an aqueous fluid andnebulized into fine droplets to be aerosolized into the lungs.

Sonic nebulizers may be used because they minimize exposing the agent toshear, which may result in degradation of the particles. Ordinarily, anaqueous aerosol is made by formulating an aqueous solution or suspensionof the particles together with conventional pharmaceutically acceptablecarriers and stabilizers. The carriers and stabilizers vary with therequirements of the particular composition, but typically includenon-ionic surfactants (TWEENS, PLURONIC®, or polyethylene glycol),innocuous proteins like serum albumin, sorbitan esters, oleic acid,lecithin, amino acids such as glycine, buffers, salts, sugars, or sugaralcohols. Aerosols generally are prepared from isotonic solutions.

The compositions and/or formulations described herein may have anysuitable osmolarity. In some embodiments, a composition and/orformulation described herein may have an osmolarity of at least about 0mOsm/L, at least about 5 mOsm/L, at least about 25 mOsm/L, at leastabout 50 mOsm/L, at least about 75 mOsm/L, at least about 100 mOsm/L, atleast about 150 mOsm/L, at least about 200 mOsm/L, at least about 250mOsm/L, or at least about 310 mOsm/L. In certain embodiments, acomposition and/or formulation described herein may have an osmolarityof less than or equal to about 310 mOsm/L, less than or equal to about250 mOsm/L, less than or equal to about 200 mOsm/L, less than or equalto about 150 mOsm/L, less than or equal to about 100 mOsm/L, less thanor equal to about 75 mOsm/L, less than or equal to about 50 mOsm/L, lessthan or equal to about 25 mOsm/L, or less than or equal to about 5mOsm/L. Combinations of the above-referenced ranges are also possible(e.g., an osmolarity of at least about 0 mOsm/L and less than or equalto about 50 mOsm/L). Other ranges are also possible. The osmolarity ofthe composition and/or formulation can be varied by changing, forexample, the concentration of salts present in the solvent of thecomposition and/or formulation.

The pharmaceutical composition of the invention may include one or morepharmaceutical agents described herein, such as a crystalline form ofCompound 3. In certain embodiments, the pharmaceutical compositionincludes a plurality of particles of the invention that comprise one ormore pharmaceutical agents in the core and/or coating of the particles.In some embodiments, the ratio of surface-altering agent topharmaceutical agent (or salt thereof) may be at least 0.001:1 (weightratio, molar ratio, or w:v ratio), at least 0.01:1, at least 0.01:1, atleast 1:1, at least 2:1, at least 3:1, at least 5:1, at least 10:1, atleast 25:1, at least 50:1, at least 100:1, or at least 500:1. In somecases, the ratio of surface-altering agent to pharmaceutical agent (orsalt thereof) may be less than or equal to 1000:1 (weight ratio or molarratio), less than or equal to 500:1, less than or equal to 100:1, lessthan or equal to 75:1, less than or equal to 50:1, less than or equal to25:1, less than or equal to 10:1, less than or equal to 5:1, less thanor equal to 3:1, less than or equal to 2:1, less than or equal to 1:1,or less than or equal to 0.1:1. Combinations of the above-referencedranges are possible (e.g., a ratio of at least 5:1 and less than orequal to 50:1). Other ranges are also possible. In some embodiments, thepharmaceutical composition of the invention includes the above-notedranges for the ratio of the weight of each one of the pharmaceuticalagents to the weight of each one of the one or more surface-alteringagents during a formation process and/or a dilution process describedherein. In certain embodiments, the pharmaceutical composition includesthe above-noted ranges for the ratio of the weight of each one of thepharmaceutical agents to the weight of each one of the one or moresurface-altering agents immediately prior to the pharmaceuticalcomposition being administered to a subject or contacted with abiological sample. The pharmaceutical agent may be present in thepharmaceutical composition of the invention in any suitable amount,e.g., at least about 0.01 wt %, at least about 0.1 wt %, at least about1 wt %, at least about 5 wt %, at least about 10 wt %, at least about 30wt % of the pharmaceutical composition. In some cases, thepharmaceutical agent may be present in the pharmaceutical composition atless than about 30 wt %, less than about 10 wt %, less than about 5 wt%, less than about 2 wt %, or less than about 1 wt % of thepharmaceutical composition. Combinations of the above-referenced rangesare also possible (e.g., present in an amount of at least about 0.1 wt %and less than about 10 wt % of the pharmaceutical composition). Otherranges are also possible. In certain embodiments, the pharmaceuticalagent is about 0.1-2 wt % of the pharmaceutical composition. In certainembodiments, the pharmaceutical agent is about 2-20 wt % of thepharmaceutical composition. In certain embodiments, the pharmaceuticalagent is about 0.2 wt %, about 0.4 wt %, about 1 wt %, about 2 wt %,about 5 wt %, or about 10 wt % of the pharmaceutical composition.

In one set of embodiments, a composition and/or formulation includes oneor more chelating agents. A chelating agent used herein refers to achemical compound that has the ability to react with a metal ion to forma complex through one or more bonds. The one or more bonds are typicallyionic or coordination bonds. The chelating agent can be an inorganic oran organic compound. A metal ion capable of catalyzing certain chemicalreactions (e.g., oxidation reactions) may lose its catalytic activitywhen the metal ion is bound to a chelating agent to form a complex.Therefore, a chelating agent may show preservative properties when itbinds to a metal ion. Any suitable chelating agent that has preservativeproperties can be used, such as phosphonic acids, aminocarboxylic acids,hydroxycarboxylic acids, polyamines, aminoalcohols, and polymericchelating agents. Specific examples of chelating agents include, but arenot limited to, ethylenediaminetetraacetic acid (EDTA), nitrilotriaceticacid (NTA), diethylenetriaminepentacetic acid (DTPA),N-hydroxyethylethylene diaminetriacetic acid (HEDTA), tetraborates,triethylamine diamine, and salts and derivatives thereof. In certainembodiments, the chelating agent is EDTA. In certain embodiments, thechelating agent is a salt of EDTA. In certain embodiments, the chelatingagent is disodium EDTA.

In certain embodiments, the pharmaceutical composition includes aplurality of particles of the invention that comprise the chelatingagent in the formulation containing the particles. In certainembodiments, the concentration of the chelating agent is greater than orequal to about 0 wt %, greater than or equal to about 0.0001 wt %,greater than or equal to about 0.003 wt %, greater than or equal toabout 0.01 wt %, greater than or equal to about 0.03 wt %, greater thanor equal to about 0.05 wt %, greater than or equal to about 0.1 wt %,greater than or equal to about 0.3 wt %, greater than or equal to about1 wt %, or greater than or equal to about 3 wt %. In certainembodiments, the concentration of the chelating agent is less than orequal to about 3 wt %, less than or equal to about 1 wt %, less than orequal to about 0.3 wt %, less than or equal to about 0.1 wt %, less thanor equal to about 0.05 wt %, less than or equal to about 0.03 wt %, lessthan or equal to about 0.01 wt %, less than or equal to about 0.003 wt%, less than or equal to about 0.001 wt %, or less than or equal toabout 0.0003 wt %. Combinations of the above-noted ranges are possible(e.g., a concentration of greater than or equal to about 0.01 wt % andless than or equal to about 0.3 wt %). Other ranges are also possible.In certain embodiments, the concentration of the chelating agent isabout 0.001-0.1 wt %. In certain embodiments, the concentration of thechelating agent is about 0.005 wt %. In certain embodiments, theconcentration of the chelating agent is about 0.01 wt %. In certainembodiments, the concentration of the chelating agent is about 0.05 wt%. In certain embodiments, the concentration of the chelating agent isabout 0.1 wt %.

In some embodiments, an antimicrobial agent may be included in acomposition and/or formulation including the coated particles describedherein. An antimicrobial agent used herein refers to a bioactive agenteffective in the inhibition of, prevention of, or protection againstmicroorganisms such as bacteria, microbes, fungi, viruses, spores,yeasts, molds, and others generally associated with infections. Examplesof antimicrobial agents include cephalosporins, clindamycin,chloramphenicol, carbapenems, minocyclines, rifampin, penicillins,monobactams, quinolones, tetracycline, macrolides, sulfa antibiotics,trimethoprim, fusidic acid, aminoglycosides, amphotericin B, azoles,flucytosine, cilofungin, bactericidal nitrofuran compounds,nanoparticles of metallic silver or an alloy of silver containing about2.5 wt % copper, silver citrate, silver acetate, silver benzoate,bismuth pyrithione, zinc pyrithione, zinc percarbonates, zincperborates, bismuth salts, parabens (e.g., methyl-, ethyl-, propyl-,butyl-, and octyl-benzoic acid esters), citric acid, benzalkoniumchloride (BAC), rifamycin, and sodium percarbonate.

In certain embodiments, the pharmaceutical composition includes aplurality of particles of the invention that comprise the antimicrobialagent in the formulation containing the particles. In certainembodiments, the concentration of the antimicrobial agent may be greaterthan or equal to about 0 wt %, greater than or equal to about 0.0001 wt%, greater than or equal to about 0.003 wt %, greater than or equal toabout 0.01 wt %, greater than or equal to about 0.03 wt %, greater thanor equal to about 0.1 wt %, greater than or equal to about 0.3 wt %,greater than or equal to about 1 wt %, or greater than or equal to about3 wt %. In certain embodiments, the concentration of the antimicrobialagent may be less than or equal to about 3 wt %, less than or equal toabout 1 wt %, less than or equal to about 0.3 wt %, less than or equalto about 0.1 wt %, less than or equal to about 0.03 wt %, less than orequal to about 0.01 wt %, less than or equal to about 0.003 wt %, lessthan or equal to about 0.001 wt %, or less than or equal to about 0.0003wt %. Combinations of the above-noted ranges are possible (e.g., aconcentration of greater than or equal to about 0.001 wt % and less thanor equal to about 0.1 wt %). Other ranges are also possible. In certainembodiments, the concentration of the antimicrobial agent is about0.001-0.05 wt %. In certain embodiments, the concentration of theantimicrobial agent is about 0.002 wt %. In certain embodiments, theconcentration of the antimicrobial agent is about 0.005 wt %. In certainembodiments, the concentration of the antimicrobial agent is about 0.01wt %. In certain embodiments, the concentration of the antimicrobialagent is about 0.02 wt %. In certain embodiments, the concentration ofthe antimicrobial agent is about 0.05 wt %.

In some embodiments, a tonicity agent may be included in a compositionand/or formulation including the coated particles described herein. Atonicity agent used herein refers to a compound or substance that can beused to adjust the composition of a formulation to the desiredosmolarity range. In certain embodiments, the desired osmolarity rangeis an isotonic range compatible with blood. In certain embodiments, thedesired osmolarity range is hypotonic. In certain embodiments, thedesired osmolarity range is hypertonic. Examples of tonicity agentsinclude glycerin, lactose, mannitol, dextrose, sodium chloride, sodiumsulfate, sorbitol, saline-sodium citrate (SSC), and the like. In certainembodiments, a combination of one or more tonicity agents may be used.In certain embodiments, the tonicity agent is glycerin. In certainembodiments, the tonicity agent is sodium chloride.

A tonicity agent (such as one described herein) may be present at asuitable concentration in a composition and/or formulation including thecoated particles described herein. In certain embodiments, theconcentration of the tonicity agent is greater than or equal to about 0wt %, greater than or equal to about 0.001 wt %, greater than or equalto about 0.03 wt %, greater than or equal to about 0.1 wt %, greaterthan or equal to about 0.3 wt %, greater than or equal to about 1 wt %,greater than or equal to about 3 wt %, greater than or equal to about 10wt %, greater than or equal to about 20 wt %, or greater than or equalto about 30 wt %. In certain embodiments, the concentration of thetonicity agent is less than or equal to about 30 wt %, less than orequal to about 10 wt %, less than or equal to about 3 wt %, less than orequal to about 1 wt %, less than or equal to about 0.3 wt %, less thanor equal to about 0.1 wt %, less than or equal to about 0.03 wt %, lessthan or equal to about 0.01 wt %, or less than or equal to about 0.003wt %. Combinations of the above-noted ranges are possible (e.g., aconcentration of greater than or equal to about 0.1 wt % and less thanor equal to about 10 wt %). Other ranges are also possible. In certainembodiments, the concentration of the tonicity agent is about 0.1-1%. Incertain embodiments, the concentration of the tonicity agent is about0.5-3%. In certain embodiments, the concentration of the tonicity agentis about 0.25 wt %. In certain embodiments, the concentration of thetonicity agent is about 0.45 wt %. In certain embodiments, theconcentration of the tonicity agent is about 0.9 wt %. In certainembodiments, the concentration of the tonicity agent is about 1.2 wt %.In certain embodiments, the concentration of the tonicity agent is about2.4 wt %. In certain embodiments, the concentration of the tonicityagent is about 5 wt %.

In some embodiments, a composition and/or formulation described hereinmay have an osmolarity of at least about 0 mOsm/L, at least about 5mOsm/L, at least about 25 mOsm/L, at least about 50 mOsm/L, at leastabout 75 mOsm/L, at least about 100 mOsm/L, at least about 150 mOsm/L,at least about 200 mOsm/L, at least about 250 mOsm/L, at least about 310mOsm/L, or at least about 450 mOsm/L. In certain embodiments, acomposition and/or formulation described herein may have an osmolarityof less than or equal to about 450 mOsm/L, less than or equal to about310 mOsm/L, less than or equal to about 250 mOsm/L, less than or equalto about 200 mOsm/L, less than or equal to about 150 mOsm/L, less thanor equal to about 100 mOsm/L, less than or equal to about 75 mOsm/L,less than or equal to about 50 mOsm/L, less than or equal to about 25mOsm/L, or less than or equal to about 5 mOsm/L. Combinations of theabove-referenced ranges are also possible (e.g., an osmolarity of atleast about 0 mOsm/L and less than or equal to about 50 mOsm/L). Otherranges are also possible.

It is appreciated in the art that the ionic strength of an inventivepharmaceutical composition that comprises a plurality of particles ofthe invention may affect the polydispersity of the plurality of theparticles. The ionic strength may also affect the colloidal stability ofthe plurality of the particles. For example, a relatively high ionicstrength of the pharmaceutical composition may cause the plurality ofparticles to coagulate and therefore may destabilize the pharmaceuticalcomposition. In some embodiments, the pharmaceutical composition isstabilized by repulsive inter-particle forces. For example, theplurality of particles may be electrically or electrostatically charged.Two charged particles may repel each other, preventing collision andaggregation. When the repulsive inter-particle forces weaken or becomeattractive, the plurality of particles may start to aggregate. Forinstance, when the ionic strength of the pharmaceutical composition isincreased to a certain level, the charges (e.g., negative charges) ofthe plurality of particles may be neutralized by the oppositely chargedions present in the pharmaceutical composition (e.g., Na⁺ ions insolution). As a result, the plurality of particles may collide and bondto each other to form aggregates (e.g., clusters or flocs) of largersizes. The formed aggregates of particles may also differ in size, andthus the polydispersity of the pharmaceutical composition may alsoincrease. For example, an inventive pharmaceutical compositioncomprising similarly-sized particles may become a pharmaceuticalcomposition comprising particles having various sizes (e.g., due toaggregation) when the ionic strength of the pharmaceutical compositionis increased beyond a certain level. In the course of aggregation, theaggregates may grow in size and eventually settle to the bottom of thecontainer, and the pharmaceutical composition is considered colloidallyunstable. Once the plurality of particles in a pharmaceuticalcomposition form aggregates, it is usually difficult to disrupt theaggregates into individual particles.

Certain pharmaceutical compositions of the invention show unexpectedproperties in that, among other things, the presence of one or moreionic tonicity agents (e.g., a salt, such as NaCl) in the pharmaceuticalcompositions at certain concentrations actually decreases or maintainsthe degree of aggregation of the particles present in the pharmaceuticalcompositions, and/or does not significantly increase aggregation. Incertain embodiments, the polydispersity of the pharmaceuticalcomposition decreases, is relatively constant, or does not change by anappreciable amount upon addition of one or more ionic tonicity agentsinto the pharmaceutical composition. For example, in some embodiments,the polydispersity of a pharmaceutical composition is relativelyconstant in the presence of added ionic strength and/or when the addedionic strength of the pharmaceutical composition is kept relativelyconstant or increased (e.g., during a formation and/or dilution processdescribed herein). In certain embodiments, when the ionic strengthincreases by at least 50%, the polydispersity increases by less thanabout 300%, less than about 100%, less than about 30%, less than about10%, less than about 3%, or less than about 1%. In certain embodiments,when the ionic strength is increased by at least 50%, the polydispersityincreases by greater than or equal to about 1%, greater than or equal toabout 3%, greater than or equal to about 10%, greater than or equal toabout 30%, or greater than or equal to about 100%. Combinations of theabove-noted ranges are possible (e.g., an increase in polydispersity ofless than 30% and greater than or equal to 3%). Other ranges are alsopossible.

The ionic strength of a pharmaceutical composition of the invention maybe controlled (e.g., increased, decreased, or maintained) through avariety of means, such as the addition of one or more ionic tonicityagents (e.g., a salt, such as NaCl) to the pharmaceutical composition.In certain embodiments, the ionic strength of a pharmaceuticalcomposition of the invention is greater than or equal to about 0.0003 M,greater than or equal to about 0.001 M, greater than or equal to about0.003 M, greater than or equal to about 0.01 M, greater than or equal toabout 0.03 M, greater than or equal to about 0.1 M, greater than orequal to about 0.3 M, greater than or equal to about 1 M, greater thanor equal to about 3 M, or greater than or equal to about 10 M. Incertain embodiments, the ionic strength of a pharmaceutical compositionof the invention is less than about 10 M, less than about 3 M, less thanabout 1 M, less than about 0.3 M, less than about 0.1 M, less than about0.03 M, less than about 0.01 M, less than about 0.003 M, less than about0.001 M, or less than about 0.0003 M. Combinations of the above-notedranges are possible (e.g., an ionic strength of greater than or equal toabout 0.01 M and less than about 1 M). Other ranges are also possible.In certain embodiments, the ionic strength of a pharmaceuticalcomposition of the invention is about 0.1 M, about 0.15 M, or about 0.3M.

In certain embodiments, the polydispersity of a pharmaceuticalcomposition does not change upon addition of one or more ionic tonicityagents into the pharmaceutical composition. In certain embodiments, thepolydispersity does not significantly increase upon addition of one ormore ionic tonicity agents into the pharmaceutical composition. Incertain embodiments, the polydispersity increases to a level describedherein upon addition of one or more ionic tonicity agents into thepharmaceutical composition.

The polydispersity of an inventive pharmaceutical composition thatcomprises a plurality of particles of the invention may be measured bythe polydispersity index (PDI). In certain embodiments, the PDI of thepharmaceutical composition is less than about 1, less than about 0.8,less than about 0.6, less than about 0.4, less than about 0.3, less thanabout 0.2, less than about 0.15, less than about 0.1, less than about0.05, less than about 0.01, or less than about 0.005. In certainembodiments, the PDI of the pharmaceutical composition is greater thanor equal to about 0.005, greater than or equal to about 0.01, greaterthan or equal to about 0.05, greater than or equal to about 0.1, greaterthan or equal to about 0.15, greater than or equal to about 0.2, greaterthan or equal to about 0.3, greater than or equal to about 0.4, greaterthan or equal to about 0.6, greater than or equal to about 0.8, orgreater than or equal to about 1. Combinations of the above-noted rangesare possible (e.g., a PDI of greater than or equal to about 0.1 and lessthan about 0.5). Other ranges are also possible. In certain embodiments,the PDI of the pharmaceutical composition is about 0.1, about 0.15, orabout 0.2. In certain embodiments, the pharmaceutical composition ishighly dispersible and does not tend to form aggregates. Even when theparticles do form aggregates, the aggregates may be easily broken upinto individual particles without rigorously agitating thepharmaceutical composition.

For example, in some embodiments, the polydispersity of a compositionand/or formulation is relatively constant in the presence of added ionicstrength and/or when the added ionic strength of the composition and/orformulation is kept relatively constant or increased (e.g., during aformation and/or dilution process). In certain embodiments, when theionic strength increases by at least 50%, the polydispersity increasesby less than or equal to about 200%, less than or equal to about 150%,less than or equal to about 100%, less than or equal to about 75%, lessthan or equal to about 50%, less than or equal to about 30%, less thanor equal to about 20%, less than or equal to about 10%, less than orequal to about 3%, or less than or equal to about 1%. In certainembodiments, when the ionic strength is increased by at least 50%, thepolydispersity increases by greater than or equal to about 1%, greaterthan or equal to about 3%, greater than or equal to about 10%, greaterthan or equal to about 30%, or greater than or equal to about 100%.Combinations of the above-noted ranges are possible (e.g., an increasein polydispersity of less than or equal to 50% and greater than or equalto 1%). Other ranges are also possible.

The ionic strength of a formulation described herein may be controlled(e.g., increased) through a variety of means, such as the addition ofone or more ionic tonicity agents (e.g., a salt such as NaCl) to theformulation. In certain embodiments, the ionic strength of a formulationdescribed herein is greater than or equal to about 0.0005 M, greaterthan or equal to about 0.001 M, greater than or equal to about 0.003 M,greater than or equal to about 0.01 M, greater than or equal to about0.03 M, greater than or equal to about 0.1 M, greater than or equal toabout 0.3 M, greater than or equal to about 1 M, greater than or equalto about 3 M, or greater than or equal to about 10 M. In certainembodiments, the ionic strength of a formulation described herein isless than or equal to about 10 M, less than or equal to about 3 M, lessthan or equal to about 1 M, less than or equal to about 0.3 M, less thanor equal to about 0.1 M, less than or equal to about 0.03 M, less thanor equal to about 0.01 M, less than or equal to about 0.003 M, less thanor equal to about 0.001 M, or less than or equal to about 0.0005 M.Combinations of the above-noted ranges are possible (e.g., an ionicstrength of greater than or equal to about 0.01 M and less than or equalto about 1 M). Other ranges are also possible. In certain embodiments,the ionic strength of a formulation described herein is about 0.1 M. Incertain embodiments, the ionic strength of a formulation describedherein is about 0.15 M. In certain embodiments, the ionic strength of aformulation described herein is about 0.3 M.

Generally, it is desired that a formulation is sterile before or uponadministration to a subject. A sterile formulation is essentially freeof pathogenic microorganisms, such as bacteria, microbes, fungi,viruses, spores, yeasts, molds, and others generally associated withinfections. In some embodiments, compositions and/or formulationsincluding the coated particles described herein may be subject to anaseptic process and/or other sterilization process. An aseptic processtypically involves sterilizing the components of a formulation, finalformulation, and/or container closure of a drug product through aprocess such as heat, gamma irradiation, ethylene oxide, or filtrationand then combining in a sterile environment. In some cases, an asepticprocess is preferred. In other embodiments, terminal sterilization ispreferred.

Examples of other sterilization methods include radiation sterilization(e.g., gamma, electron, or x-ray radiation), heat sterilization, sterilefiltration, and ethylene oxide sterilization. The terms “radiation” and“irradiation” are used herein interchangeably. Unlike othersterilization methods, radiation sterilization has the advantage of highpenetrating ability and instantaneous effects, without the need tocontrol temperature, pressure, vacuum, or humidity in some instances. Incertain embodiments, the radiation used to sterilize the coatedparticles described herein is gamma radiation. Gamma radiation may beapplied in an amount sufficient to kill most or substantially all of themicrobes in or on the coated particles. The temperature of the coatedparticles described herein and the rate of radiation may be relativelyconstant during the entire gamma radiation period. Gamma irradiation maybe performed at any suitable temperature (e.g., ambient temperature,about 40° C., between about 30 to about 50° C.). Unless otherwiseindicated, measurements of gamma irradiation described herein refer toones performed at about 40° C.

In embodiments in which a sterilization process is used, it may bedesired that the process does not: (1) significantly change the particlesize of the coated particles described herein; (2) significantly changethe integrity of the active ingredient (such as a drug) of the coatedparticles described herein; and (3) generate unacceptable concentrationsof impurities during or following the process. In certain embodiments,the impurities generated during or following the process are degradantsof the active ingredient of the coated particles described herein.

In certain embodiments, a process used to sterilize a composition and/orformulation described herein results in the presence of one or moredegradants in the formulation at less than or equal to about 10 wt %(relative to the weight of the undegraded drug), less than or equal toabout 3 wt %, less than or equal to about 2 wt %, less than or equal toabout 1.5 wt %, less than or equal to about 1 wt %, less than or equalto about 0.9 wt %, less than or equal to about 0.8 wt %, less than orequal to about 0.7 wt %, less than or equal to about 0.6 wt %, less thanor equal to about 0.5 wt %, less than or equal to about 0.4 wt %, lessthan or equal to about 0.3 wt %, less than or equal to about 0.2 wt %,less than or equal to about 0.15 wt %, less than or equal to about 0.1wt %, less than or equal to about 0.03 wt %, less than or equal to about0.01 wt %, less than or equal to about 0.003 wt %, or less than or equalto about 0.001 wt %. In some embodiments, the process results in adegradant in the formulation at greater than or equal to about 0.001 wt%, greater than or equal to about 0.003 wt %, greater than or equal toabout 0.01 wt %, greater than or equal to about 0.03 wt %, greater thanor equal to about 0.1 wt %, greater than or equal to about 0.3 wt %,greater than or equal to about 1 wt %, greater than or equal to about 3wt %, or greater than or equal to about 10 wt %. Combinations of theabove-referenced ranges are also possible (e.g., less than or equal toabout 1 wt % and greater than or equal to about 0.01 wt %). Other rangesare also possible.

When gamma irradiation is used in a sterilization process, thecumulative amount of the gamma radiation used may vary. In certainembodiments, the cumulative amount of the gamma radiation is greaterthan or equal to about 0.1 kGy, greater than or equal to about 0.3 kGy,greater than or equal to about 1 kGy, greater than or equal to about 3kGy, greater than or equal to about 10 kGy, greater than or equal toabout 30 kGy, greater than or equal to about 100 kGy, or greater than orequal to about 300 kGy. In certain embodiments, the cumulative amount ofthe gamma radiation is less than or equal to about 0.1 kGy, less than orequal to about 0.3 kGy, less than or equal to about 1 kGy, less than orequal to about 3 kGy, less than or equal to about 10 kGy, less than orequal to about 30 kGy, less than or equal to about 100 kGy, or less thanor equal to about 300 kGy. Combinations of the above-noted ranges arepossible (e.g., greater than or equal to about 1 kGy and less than orequal to about 30 kGy). Other ranges are also possible. In certainembodiments, multiple doses of radiation are utilized to achieve adesired cumulative radiation dosage.

The compositions and/or formulations described herein may have anysuitable pH values. The term “pH,” unless otherwise provided, refers topH measured at ambient temperature (e.g., about 20° C., about 23° C., orabout 25° C.). The compositions and/or formulations have, for example,an acidic pH, a neutral pH, or a basic pH and may depend on, forexample, where the compositions and/or formulations are to be deliveredin the body. In certain embodiments, the compositions and/orformulations have a physiological pH. In certain embodiments, the pHvalue of the compositions and/or formulations is at least about 1, atleast about 2, at least about 3, at least about 4, at least about 5, atleast about 6, at least about 6.2, at least about 6.4, at least about6.6, at least about 6.8, at least about 7, at least about 7.2, at leastabout 7.4, at least about 7.6, at least about 7.8, at least about 8, atleast about 8.2, at least about 8.4, at least about 8.6, at least about8.8, at least about 9, at least about 10, at least about 11, or at leastabout 12. In certain embodiments, the pH value of the compositionsand/or formulations is less than or equal to about 12, less than orequal to about 11, less than or equal to about 10, less than or equal toabout 9, less than or equal to about 8.8, less than or equal to about8.6, less than or equal to about 8.4, less than or equal to about 8.2,less than or equal to about 8, less than or equal to about 7.8, lessthan or equal to about 7.6, less than or equal to about 7.4, less thanor equal to about 7.2, less than or equal to about 7, less than or equalto about 6.8, less than or equal to about 6.6, less than or equal toabout 6.4, less than or equal to about 6.2, less than or equal to about6, less than or equal to about 5, less than or equal to about 4, lessthan or equal to about 3, less than or equal to about 2, or less than orequal to about 1. Combinations of the above-noted ranges are possible(e.g., a pH value of at least about 5 and less than or equal to about8.2). Other ranges are also possible. In certain embodiments, the pHvalue of the compositions and/or formulations described herein is atleast about 5 and less than or equal to about 8.

In some embodiments, the particles, compositions, and/or formulationsdescribed herein increase the ocular bioavailability of Compound 3 by atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 100%, at least about150%, at least about 200%, at least about 5 fold, at least about 10fold, at least about 20 fold, at least about 50 fold, at least about 100fold, at least about 500 fold, or at least about 1000 fold. In certainembodiments the particles, compositions, and/or formulations describedherein increase the ocular bioavailability of Compound 3 by less than orequal to about 1000 fold, less than or equal to about 500 fold, lessthan or equal to about 100 fold, less than or equal to about 50 fold,less than or equal to about 20 fold, less than or equal to about 10fold, less than or equal to about 5 fold, less than or equal to about200%, less than or equal to about 150%, less than or equal to about100%, less than or equal to about 90%, less than or equal to about 80%,less than or equal to about 70%, less than or equal to about 60%, lessthan or equal to about 50%, less than or equal to about 40%, less thanor equal to about 30%, less than or equal to about 20%, or less than orequal to about 10%. Combinations of the above-referenced ranges are alsopossible (e.g., an increase of at least about 10% and less than or equalto about 10 fold). Other ranges are also possible. In some instances,the AUC of Compound 3 increases at a tissue and/or fluid in the front ofthe eye. In other instances, the AUC of Compound 3 increases at a tissueand/or fluid in the back of the eye.

In general, an increase in ocular bioavailability may be calculated bytaking the difference in the AUC measured in an ocular tissue ofinterest (e.g., in aqueous humor) between those of a test compositionand a control composition, and dividing the difference by thebioavailability of the control composition. A test composition mayinclude particles comprising a crystalline form of Compound 3, and theparticles may be characterized as being mucus penetrating (e.g., havinga relative velocity in mucus of greater than about 0.5, or another otherrelative velocity described herein). A control composition may includeparticles comprising the same crystalline form of Compound 3 as thatpresent in the test composition, the particles having a substantiallysimilar size as those of the test composition, but which are not mucuspenetrating (e.g., having a relative velocity in mucus of less than orequal to about 0.5, or another other relative velocity describedherein).

Ocular bioavailability of Compound 3 may be measured in an appropriateanimal model (e.g. in a New Zealand white rabbit model, or a Gottingenmini-pig model). The concentration of Compound 3 and, when appropriate,its metabolite(s), in appropriate ocular tissues or fluids is measuredas a function of time after administration. Other methods of measuringocular bioavailability of Compound 3 are possible.

In some embodiments, the concentration of Compound 3 in an ocular tissueand/or fluid may be increased when the crystalline form of Compound 3 isdelivered (e.g., via topical administration to the eye) using theparticles, compositions, and/or formulations described herein comparedto when the crystalline form of Compound 3 is delivered using certainexisting particles, compositions, and/or formulations that contain thesame the crystalline form of Compound 3 (or compared to the delivery ofthe same crystalline form of Compound 3 (e.g., of similar size) as thecoated particle in question, but which does not include the coating). Incertain embodiments, a dose of the particles, compositions, and/orformulations is administered, followed by the measurement of theconcentration of the crystalline form of Compound 3 in a tissue and/orfluid of the eye. For purposes of comparison, the amount of thecrystalline form of Compound 3 included in the administered dose of theparticles, compositions, and/or formulations described herein may besimilar or substantially equal to the amount of the crystalline form ofCompound 3 included in the administered dose of the existing particles,compositions, and/or formulations. In certain embodiments, theconcentration of Compound 3 in a tissue and/or fluid of the eye ismeasured at a certain time subsequent to the administration (“timepost-dose”) of a dose of the particles, compositions, and/orformulations described herein or of the existing particles,compositions, and/or formulations. In certain embodiments, the time whenthe concentration is measured is about 1 min, about 10 min, about 30min, about 1 h, about 2 h, about 3 h, about 4 h, about 5 h, about 6 h,about 7 h, about 8 h, about 9 h, about 10 h, about 11 h, about 12 h,about 18 h, about 24 h, about 36 h, or about 48 h, post-dose.

In some embodiments, the concentration of Compound 3 in a tissue and/orfluid may increase due to, at least in part, a coating on core particlescomprising the crystalline form of Compound 3 that renders the particlesmucus penetrating, compared to particles of the same crystalline form ofCompound 3 (e.g., of similar size) as the coated particle in question,but which does not include the coating. In some embodiments, theparticles, compositions, and/or formulations described herein increasesthe concentration of Compound 3 in a tissue and/or fluid by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100%, at least about 200%,at least about 300%, at least about 400%, at least about 500%, or atleast about 10 fold, at least about 20 fold, at least about 50 fold, atleast about 100 fold, at least about 1000 fold, at least about 10⁴ fold,at least about 10⁵ fold, or at least about 10⁶ fold. In some cases, theparticles, compositions, and/or formulations described herein increasesthe concentration of Compound 3 in a tissue and/or fluid by less than orequal to about 10⁶ fold, less than or equal to about 10⁵ fold, less thanor equal to about 10⁴ fold, 1000 fold, less than or equal to about 100fold, less than or equal to about 10 fold, less than or equal to about500%, less than or equal to about 400%, less than or equal to about300%, less than or equal to about 200%, less than or equal to about100%, less than or equal to about 90%, less than or equal to about 80%,less than or equal to about 70%, less than or equal to about 60%, lessthan or equal to about 50%, less than or equal to about 40%, less thanor equal to about 30%, less than or equal to about 20%, or less than orequal to about 10%. Combinations of the above-referenced ranges are alsopossible (e.g., an increase of greater than or equal to about 10% andless than or equal to about 90%). Other ranges are also possible. Insome instances, the concentration of Compound 3 increases at a tissueand/or fluid in the front of the eye. In other instances, theconcentration of Compound 3 increases at a tissue and/or fluid in theback of the eye.

The ocular concentration of Compound 3, and, when appropriate, itsmetabolite(s), in appropriate ocular fluids or tissues may be measuredas a function of time in vivo using an appropriate animal model. Onemethod of determining the ocular concentration of Compound 3 involvesdissecting of the eye to isolate tissues of interest (e.g., in an animalmodel comparable to the subject). The concentration of Compound 3 in thetissues of interest is then determined by HPLC or LC/MS analysis.

In certain embodiments, the period of time between administration of theparticles described herein and obtaining a sample for measurement ofconcentration or AUC is less than about 1 hour, less than or equal toabout 2 hours, less than or equal to about 3 hours, less than or equalto about 4 hours, less than or equal to about 6 hours, less than orequal to about 12 hours, less than or equal to about 36 hours, or lessthan or equal to about 48 hours. In certain embodiments, the period oftime is at least about 1 hour, at least about 2 hours, at least about 3hours, at least about 4 hours, at least about 6 hours, at least about 8hours, at least about 12 hours, at least about 36 hours, or at leastabout 48 hours. Combinations of the above-referenced ranges are alsopossible (e.g., a period of time between consecutive doses of greaterthan or equal to about 3 hours and less than or equal to about 12hours). Other ranges are also possible.

Other methods of measuring the concentration of Compound 3 in an eye ofa subject or an animal model are also possible. In some embodiments, theconcentration of Compound 3 may be measured in the eye of the subjectdirectly or indirectly (e.g., taking a sample of fluid, such as vitreoushumor, from an eye of the subject).

In general, an increase in concentration of Compound 3 in an ocular sitemay be calculated by taking the difference in concentration measuredbetween those of a test composition and a control composition, anddividing the difference by the concentration of the control composition.A test composition may include particles comprising a crystalline formof Compound 3, and the particles may be characterized as being mucuspenetrating (e.g., having a relative velocity of greater than about 0.5,or another other relative velocity described herein). A controlcomposition may include particles comprising the same crystalline formof Compound 3 as that present in the test composition, the particleshaving a substantially similar size as those of the test composition,but which are not mucus penetrating (e.g., having a relative velocity ofless than about 0.5, or another other relative velocity describedherein).

As described herein, in some embodiments, the particles, compositions,and/or formulations described herein, or a component thereof, is presentin a sufficient amount to increase the bioavailability and/orconcentration of Compound 3 in an ocular tissue, compared to thecrystalline form of Compound 3 administered to the ocular tissue in theabsence of the particles, compositions, and formulations describedherein, or a component thereof.

The ocular tissue may be an anterior ocular tissue (e.g., a palpebralconjunctiva, a bulbar conjunctiva, or a cornea). In certain embodiments,the core particle of a formulation comprising a crystalline form ofCompound 3 is present in a sufficient amount to increase thebioavailability and/or concentration of Compound 3 in an ocular tissue.In certain embodiments, the coating on the core particle of aformulation comprising a crystalline form of Compound 3 is present in asufficient amount to increase the bioavailability and/or concentrationof Compound 3 in an ocular tissue. In certain embodiments, the coatingon the core particle of a formulation comprising a crystalline form ofCompound 3 is present in a sufficient amount to increase theconcentration of Compound 3 in an ocular tissue after at least 10minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, atleast 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, atleast 9 hours, at least 12 hours, at least 18 hours, or at least 24hours after administration of the formulation to the ocular tissue. Incertain embodiments, the coating on the core particle of a formulationcomprising a crystalline form of Compound 3 is present in a sufficientamount to increase the concentration of Compound 3 in an ocular tissueafter less than or equal to 24 hours, less than or equal to 18 hours,less than or equal to 12 hours, less than or equal to 9 hours, less thanor equal to 6 hours, less than or equal to 4 hours, less than or equalto 3 hours, less than or equal to 2 hours, less than or equal to 1 hour,less than or equal to 30 minutes, less than or equal to 20 minutes, orless than or equal to 10 minutes after administration of the formulationto the ocular tissue. Combinations of the above-referenced ranges arealso possible (e.g., the concentration of Compound 3 increases after atleast 10 minutes and less than or equal to 2 hours). Other ranges arealso possible. In certain embodiments, the coating on the core particleof a formulation comprising a crystalline form of Compound 3 is presentin a sufficient amount to increase the concentration of Compound 3 in anocular tissue after about 30 minutes after administration of theformulation to the ocular tissue.

In some embodiments, the particles, compositions, and/or formulationsdescribed herein can be administered topically to an eye of a subject invarious forms of doses. For example, the particles, compositions, and/orformulations described herein may be administered in a single unit doseor repeatedly administered in a plurality of single unit doses. A unitdose is a discrete amount of the particles, compositions, and/orformulations described herein comprising a predetermined amount of apharmaceutical agent. In some embodiments, fewer numbers of doses (e.g.,½, ⅓, or ¼ the number doses) are required using the particles describedherein having a mucus-penetrating coating compared to particles that donot have such a coating.

The exact amount of the particles, compositions, and/or formulationsdescribed herein required to achieve a therapeutically orprophylactically effective amount will vary from subject to subject,depending, for example, on species, age, and general condition of asubject, severity of the side effects or disorder, mode ofadministration, and the like. The particles, compositions, and/orformulations described herein can be delivered using repeatedadministrations where there is a period of time between consecutivedoses. Repeated administration may be advantageous because it may allowthe eye to be exposed to a therapeutically or prophylactically effectiveamount of Compound 3 for a period of time that is sufficiently long forthe ocular condition to be treated, prevented, or managed. In certainembodiments, the period of time between consecutive doses is less thanor equal to about 1 hour, less than or equal to about 2 hours, less thanor equal to about 3 hours, less than or equal to about 4 hours, lessthan or equal to about 6 hours, less than or equal to about 12 hours,less than or equal to about 36 hours, or less than or equal to about 48hours. In certain embodiments, the period of time between consecutivedoses is at least about 1 hour, at least about 2 hours, at least about 3hours, at least about 4 hours, at least about 6 hours, at least about 12hours, at least about 36 hours, or at least about 48 hours. Combinationsof the above-referenced ranges are also possible (e.g., a period of timebetween consecutive doses of greater than or equal to about 3 hours andless than or equal to about 12 hours). Other ranges are also possible.

Delivery of the particles, compositions, and/or formulations describedherein to an ocular tissue may result in ophthalmically efficaciouslevels of Compound 3 in the ocular tissue for an extended period of timeafter administration (e.g., topical administration or administration bydirect injection). An ophthalmically efficacious level of Compound 3refers to an amount sufficient to elicit the desired biological responseof an ocular tissue, i.e., treating an ocular disease. As will beappreciated by those skilled in this art, the ophthalmically efficaciouslevel of Compound 3 may vary depending on such factors as the desiredbiological endpoint, the pharmacokinetics of Compound 3, the oculardisease being treated, the mode of administration, and the age andhealth of the subject. In certain embodiments, the ophthalmicallyefficacious level of Compound 3 is an amount of Compound 3, alone or incombination with other therapies, which provides a therapeutic benefitin the treatment of the ocular condition. The ophthalmically efficaciouslevel of Compound 3 can encompass a level that improves overall therapy,reduces or avoids symptoms or causes of the ocular condition, orenhances the therapeutic efficacy of another therapeutic agent.

In some embodiments, an ophthalmically efficacious level of Compound 3may be gauged, at least in part, by the maximum concentration (C_(max))of Compound 3 in the ocular tissue after administration.

In some embodiments, the ophthalmically efficacious levels of Compound 3are gauged, at least in part, by minimally efficacious concentrations ofCompound 3, e.g., IC₅₀ or IC₉₀, as known in the art.

In certain embodiments in which ophthalmically efficacious levels (orC_(max), IC₅₀, or IC₉₀) of Compound 3 are present in the ocular tissuefor an extended period of time after administration, the extended periodof time after administration can range from hours to days. In certainembodiments, the extended period of time after administration is atleast 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, atleast 9 hours, at least 12 hours, at least 1 day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, or atleast 1 week. In certain embodiments, the extended period of time afteradministration is less than or equal to 1 week, less than or equal to 6days, less than or equal to 5 days, less than or equal to 4 days, lessthan or equal to 3 days, less than or equal to 2 days, less than orequal to 1 day, less than or equal to 12 hours, less than or equal to 9hours, less than or equal to 6 hours, less than or equal to 4 hours,less than or equal to 2 hours, less than or equal to 1 hour.Combinations of the above-referenced ranges are also possible (e.g., anextended period of time of at least about 4 hours and less than or equalto about 1 week). Other ranges are also possible.

In certain embodiments, the particles, compositions, and/or formulationsdescribed herein may be at dosage levels sufficient to deliver aneffective amount of Compound 3 to an eye of a subject to obtain adesired therapeutic or prophylactic effect. In certain embodiments, aneffective amount of Compound 3 that is delivered to an appropriate eyetissue is at least about 10⁻³ ng/g, at least about 10⁻² ng/g, at leastabout 10⁻¹ ng/g, at least about 1 ng/g, at least about 10¹ ng/g, atleast about 10² ng/g, at least about 10³ ng/g, at least about 10⁴ ng/g,at least about 10⁵ ng/g, or at least about 10⁶ ng/g of tissue weight. Incertain embodiments, an effective amount of Compound 3 that is deliveredto the eye is less than or equal to about 10⁶ ng/g, less than or equalto about 10⁵ ng/g, less than or equal to about 10⁴ ng/g, less than orequal to about 10³ ng/g, less than or equal to about 10² ng/g, less thanor equal to about 10¹ ng/g, less than or equal to about 1 ng/g, lessthan or equal to about 10⁻¹ ng/g, less than or equal to about 10⁻² ng/g,or less than or equal to about 10⁻³ ng/g of tissue weight. Combinationsof the above-referenced ranges are also possible (e.g., an effectiveamount of Compound 3 of at least about 10⁻² ng/g and less than or equalto about 10³ ng/g of tissue weight). Other ranges are also possible. Incertain embodiments, the particles, compositions, and/or formulationsdescribed herein may be at dosage levels sufficient to deliver aneffective amount of Compound 3 to the back of an eye of a subject toobtain a desired therapeutic or prophylactic effect.

It will be appreciated that dose ranges as described herein provideguidance for the administration of provided particles, compositions,and/or formulations to an adult. The amount to be administered to, forexample, a child or an adolescent can be determined by a medicalpractitioner or person skilled in the art and can be lower or the sameas that administered to an adult.

The particles, compositions, and/or formulations described herein may betopically administered (e.g., ocular or dermal) by any method, forexample, as by drops, powders, ointments, or creams. Other topicaladministration approaches or forms are also possible.

In certain embodiments, the compositions and/or formulations describedherein are packaged as a ready to use shelf stable suspension. Eye dropformulations are traditionally liquid formulations (solutions orsuspensions) which can be packaged in dropper bottles (which dispense astandard drop volume of liquid) or in individual use droppers (typicallyused for preservative free drops; used once and disposed). They can bestored in suspension and may retain the characteristics which allow theparticles to avoid adhesion to mucus.

Methods of Preparing Particles and Pharmaceutical Compositions Thereof

In one aspect, the present invention provides methods of preparing theparticles of the invention. Methods of preparing similar particles havebeen described in U.S. Patent Publication Nos. 2013/0316001,2013/0316006, 2013/0316009, and 20130323179, each of which isincorporated by reference herein in its entirety.

The core of the particle may be formed by any suitable method. Suitablemethods may include, for example, top-down techniques, i.e. techniquesbased on size reduction of relatively large particles into smallerparticles (e.g., milling or homogenization) or bottom-up techniques,i.e. techniques based on the growth of particles from smaller particlesor individual molecules (e.g., precipitation or spray-freezing intoliquid).

In some embodiments, the core of the particle may be coated with acoating. For example, the core may be provided or formed in a firststep, and then the core may be coated in a second step. In someembodiments, the core particle is formed and coated substantiallysimultaneously (e.g., in a single step).

In some embodiments, the particle is formed by a method that involvesusing a formulation process, a milling process, and/or a dilutionprocess. In certain embodiments, a method of forming the particleincludes a milling process, optionally with a formulation process and/ora dilution process. A formulation process may be used to form asuspension comprising a core material, one or more surface-alteringagents, and other components, such as solvents, tonicity agents,chelating agents, salts, and/or buffers (e.g., a sodium citrate andcitric acid buffer), each of which is as described herein. Theformulation process may be performed using a formulation vessel. Thecore material and other components may be added into the formulationvessel at the same time or different times. A mixture of the corematerial and/or one or more other components may be stirred and/orshaken, or otherwise agitated in the vessel to facilitate suspending thecomponents to form the suspension. The temperature and/or pressure ofthe core material, other components, and/or mixture may also beindividually increased or decreased to facilitate the suspendingprocess. In some embodiments, the core material and other components areprocessed as described herein in the formulation vessel under an inertatmosphere (e.g., nitrogen or argon) and/or protected from light. Thesuspension obtained from the formulation vessel may be subsequentlysubject to a milling process which may be followed by a dilutionprocess.

In some embodiments involving a core comprising a solid material (e.g.,crystalline compound of the invention) a milling process may be used toreduce the size of the solid material to form particles in a micrometerto nanometer size range. The milling process may be performed using amill or other suitable apparatus. Dry and wet milling processes such asjet milling, cryo-milling, ball milling, media milling, sonication, andhomogenization are known and can be used in methods of the invention.For example, in a wet milling process, a suspension of the solidmaterial to be used to form the core (“core material”) is agitated withor without excipients to reduce the size of the core to be formed. Drymilling is a process wherein the core material is mixed with millingmedia with or without excipients to reduce the size of the core to beformed. In a cryo-milling process, a suspension of the core material ismixed with milling media with or without excipients under cooledtemperatures. In certain embodiments, when surface-altering agents areemployed, a suspension comprising coated particles is obtained from themilling process. In certain embodiments, when surface-altering agentsare not employed, a suspension comprising uncoated particles is obtainedfrom the milling process.

The suspension of particles (coated or uncoated) of the inventionobtained from a milling process may be further processed with a dilutionprocess. A dilution process may be used to achieve a target dosingconcentration by diluting a suspension of particles that were formedduring a milling process, with or without surface-altering agents and/orother components. In certain embodiments, when a suspension of coatedparticles that comprise a first surface-altering agent is processed witha dilution process involving a second surface-altering agent, asuspension of coated particles that comprise the second surface-alteringagent is obtained from the dilution process. In certain embodiments,when a suspension of coated particles that comprise a surface-alteringagent is processed with a dilution process involving no or the samesurface-altering agent, a suspension of coated particles that comprisethe surface-altering agent is obtained from the dilution process. Incertain embodiments, when a suspension of uncoated particles isprocessed with a dilution process involving a surface-altering agent, asuspension of coated particles comprising the surface-altering agent isobtained from the dilution process. The dilution process may beperformed using a product vessel or any other suitable apparatus. Incertain embodiments, the suspension of the particles is diluted, i.e.,mixed or otherwise processed with a diluent, in the product vessel. Thediluent may contain solvents, surface-altering agents, tonicity agents,chelating agents, salts, anti-microbial agents or a combination thereof,as described herein. The suspension and the diluent may be added intothe product vessel at the same time or different times. In certainembodiments when the suspension is obtained from a milling processinvolving milling media, the milling media may be separated from thesuspension before the suspension is added into the product vessel. Thesuspension, the diluent, or the mixture of the suspension and thediluent may be stirred and/or shaken, or otherwise agitated, to form theparticles and/or pharmaceutical compositions of the invention. Thetemperature and/or pressure of the suspension, the diluent, or themixture may also be individually increased or decreased to form thecoated particles. In some embodiments, the suspension and the diluentare processed in the product vessel under an inert atmosphere (e.g.,nitrogen or argon) and/or protected from light.

In some embodiments, the core and/or coated particles may be produced bymilling of a solid material (e.g., a pharmaceutical agent) in thepresence of one or more surface-altering agents. Small particles of asolid material may require the presence of one or more surface-alteringagents, which may function as a stabilizer in some embodiments, in orderto stabilize a suspension of particles without agglomeration oraggregation in a liquid solution. In some such embodiments, thestabilizer may act as a surface-altering agent, forming the coatedparticles of the invention.

As described herein, a method of forming the core and/or the coatedparticles, may involve choosing a surface-altering agent that issuitable for both milling and forming a coating on the core, wherein thecoating renders the particle mucus penetrating.

In a wet milling process, milling may be performed in a dispersion(e.g., an aqueous dispersion) containing at least one surface-alteringagent, a grinding medium, a solid to be milled (e.g., a solidpharmaceutical agent), and a solvent. The solvent described hereinincludes a single solvent or a mixture of different solvents. Anysuitable amount of a surface-altering agent can be included in thesolvent. In some embodiments, the surface-altering agent may be presentin the solvent in an amount of at least about 0.001% (wt % or % weightto volume (w:v)), at least about 0.01%, at least about 0.1%, at leastabout 1%, at least about 3%, at least about 10%, at least about 30%, orat least about 60% of the solvent. In some cases, the surface-alteringagent may be present in the solvent in an amount of about 100% (e.g., inan instance where the surface-altering agent is the solvent). In otherembodiments, the surface-altering agent may be present in the solvent inan amount of less than about 100%, less than about 60%, less than about30%, less than about 10%, less than about 3%, or less than about 1% ofthe solvent. Combinations of the above-referenced ranges are alsopossible (e.g., an amount of less than about 3% and at least about 1% ofthe solvent). Other ranges are also possible. In certain embodiments,the surface-altering agent is present in the solvent in an amount ofabout 0.01-2%, about 0.2-20%, about 0.1%, about 0.4%, about 1%, about2%, about 5%, or about 10% of the solvent.

The particular range chosen may influence factors that may affect theability of the particles to penetrate mucus such as the stability of thecoating of the surface-altering agent on the particle surface, theaverage thickness of the coating of the surface-altering agent on theparticles, the orientation of the surface-altering agent on theparticles, the density of the surface altering agent on the particles,the ratio of the surface-altering agent to pharmaceutical agent, theconcentration of the pharmaceutical agent, the size, dispersibility, andpolydispersity of the particles formed, and the morphology of theparticles formed.

The pharmaceutical agent may be present in the solvent in any suitableamount. In some embodiments, the pharmaceutical agent is present in anamount of at least about 0.001% (wt % or % weight to volume (w:v)), atleast about 0.01%, at least about 0.1%, at least about 1%, at leastabout 3%, at least about 10%, at least about 30%, or at least about 60%of the solvent. In some cases, the pharmaceutical agent may be presentin the solvent in an amount of less than about 100%, less than about60%, less than about 30%, less than about 10%, less than about 3%, orless than about 1% of the solvent. Combinations of the above-referencedranges are also possible (e.g., an amount of less than about 30% and atleast about 1% of the solvent).

The ratio of surface-altering agent to pharmaceutical agent in a solventmay also vary. In some embodiments, the ratio of the surface-alteringagent to pharmaceutical agent is at least about 0.001:1 (weight ratio,molar ratio, or w:v), at least about 0.01:1, at least about 0.01:1, atleast about 1:1, at least about 2:1, at least about 3:1, at least about5:1, at least about 10:1, at least about 30:1, at least about 100:1, orat least about 1000:1. In some embodiments, the ratio of thesurface-altering agent to pharmaceutical agent is less than 1000:1(weight ratio, molar ratio, or w:v), less than about 100:1, less thanabout 30:1, less than about 10:1, less than about 5:1, less than about3:1, less than about 2:1, less than about 1:1, or less than about 0.1:1.Combinations of the above-referenced ranges are possible (e.g., a ratioof at least about 5:1 and less than about 30:1). Other ranges are alsopossible.

The surface-altering agents described herein that may act as stabilizersmay be, for example, polymers or surfactants. Examples of polymersinclude those suitable for use in the coating of the particles of theinvention, such as poly(vinyl alcohol) and PLURONICS®. Examples ofsurfactants include L-α-phosphatidylcholine (PC),1,2-dipalmitoylphosphatidycholine (DPPC), oleic acid, sorbitantrioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxylenesorbitan fatty acid esters (TWEENS), polysorbates (e.g., polyoxyethylenesorbitan monooleate) (e.g., TWEEN 80®), polyoxyethylene sorbitanmonostearate (e.g., TWEEN 60®), polyoxyethylene sorbitan monopalmitate(e.g., TWEEN 40®), polyoxyethylene sorbitan monolaurate (e.g., TWEEN20®), natural lecithin, oleyl polyoxyethylene ether, stearylpolyoxyethylene ether, lauryl polyoxyethylene ether, polyoxylene alkylethers, block copolymers of oxyethylene and oxypropylene,polyoxyethylene stearates, polyoxyethylene castor oil and theirderivatives, Vitamin-PEG and their derivatives, synthetic lecithin,diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate,isopropyl myristate, glyceryl monooleate, glyceryl monostearate,glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, polyethyleneglycol, cetyl pyridinium chloride, benzalkonium chloride, olive oil,glyceryl monolaurate, corn oil, cotton seed oil, and sunflower seed oil.Derivatives of the above-noted compounds are also possible. Combinationsof the above-noted compounds and others described herein may also beused as surface-altering agents in the inventive particles. As describedherein, in some embodiments a surface-altering agent may act as astabilizer, a surfactant, and/or an emulsifier. In some embodiments, thesurface altering agent may aid particle transport in mucus.

A stabilizer used for milling may form the coating of a particle of theinvention, wherein the coating renders the particle mucus penetrating.The stabilizer may also be exchanged with one or more othersurface-altering agents after the particle has been formed. For example,a first stabilizer/surface-altering agent may be used during a millingprocess and may form a first coating of the particle of the invention,and all or part of the first stabilizer/surface-altering agent may thenbe exchanged with a second stabilizer/surface-altering agent to form asecond coating of the particle. In some embodiments, the secondstabilizer/surface-altering agent may render the particle mucuspenetrating more than the first stabilizer/surface-altering agent. Insome embodiments, a particle comprising multiple coatings that includemultiple surface-altering agents is formed by a method of the invention.

Any suitable grinding medium can be used for milling. In someembodiments, a ceramic and/or polymeric material and/or a metal can beused. Examples of suitable materials include zirconium oxide, siliconcarbide, silicon oxide, silicon nitride, zirconium silicate, yttriumoxide, glass, alumina, alpha-alumina, aluminum oxide, polystyrene,poly(methyl methacrylate), titanium, and steel. A grinding medium mayhave any suitable size. For example, the grinding medium may have anaverage diameter of at least about 0.1 mm, at least about 0.2 mm, atleast about 0.5 mm, at least about 0.8 mm, at least about 1 mm, at leastabout 2 mm, or at least about 5 mm. In some cases, the grinding mediummay have an average diameter of less than about 5 mm, less than about 2mm, less than about 1 mm, less than about 0.8, less than about 0.5 mm,or less than about 0.2 mm. Combinations of the above-referenced rangesare also possible (e.g., an average diameter of at least about 0.5millimeters and less than about 1 mm). Other ranges are also possible.

A solvent may be used for milling. The choice of the solvent suitablefor milling may depend on factors like the solid material (e.g., a solidpharmaceutical agent) being milled, the particular type ofstabilizer/surface-altering agent (e.g., one that may render theparticle mucus penetrating), and the grinding material. The solventsuitable for milling may be one of those solvents that do notsubstantially dissolve the solid material or the grinding material, butdissolve the stabilizer/surface-altering agent to a suitable degree.Examples of the solvents suitable for milling include water, aqueoussolutions, buffered solutions, alcohols (e.g., ethanol, methanol, andbutanol), and mixtures thereof, each of which may optionally includeother components, such as one or more pharmaceutical excipients,polymers, pharmaceutical agents, salts, preservative agents, viscositymodifiers, tonicity modifiers, taste masking agents, antioxidants, andpH modifiers. In some embodiments, the solvent suitable for milling isan organic solvent.

A pharmaceutical agent described herein (e.g., a crystalline form ofCompound 3) may have a suitable solubility in a solvent suitable formilling, such as a solubility in one or more ranges described herein foraqueous solubility or for solubility in a coating solution. Apharmaceutical agent having a relatively low solubility in a solvent(e.g., water or a coating solution) may be preferred because a millingprocess described herein typically requires a material (e.g., apharmaceutical agent) to be in a solid form in order for the material tobe milled. In some cases, if the material to be milled has a relativelyhigh soluble in a solvent (e.g., water or a coating solution) used inthe milling process, milling may not be conducted because significant orcomplete dissolution of the material to be milled in the solvent willoccur. In certain embodiments, a relatively high solubility of a solidmaterial (e.g., a solid pharmaceutical agent) in a solvent is at leastabout 1 mg/mL, at least about 3 mg/mL, or at least about 10 mg/mL at 25°C. In certain embodiments, a relatively low solubility of a substance(e.g., a pharmaceutical agent) in a solvent is less than about 1 mg/mL,less than about 0.3 mg/mL, less than about 0.1 mg/mL, less than about0.03 mg/mL, less than about 0.01 mg/mL, less than about 0.003 mg/mL, orless than about 0.001 mg/mL at 25° C. The solid material may have theseor other ranges of solubilities at any point throughout the pH range(e.g., from pH 1 to pH 14).

In other embodiments, the core and/or coated particles may be formed byan emulsification process or technique (emulsification) known in theart. See, e.g., U.S. Patent Publication No. 20130316006. Generally,emulsification techniques may involve dissolving or dispersing amaterial to be used as the core in a solvent; this solution ordispersion is then emulsified in a second immiscible solvent, therebyforming a plurality of particles comprising the material. Suitableemulsification techniques may include formation of oil-in-wateremulsions, water-in-oil emulsions, water-oil-water emulsions,oil-water-oil emulsions, solid-in-oil-in-water emulsions, andsolid-in-water-in-oil emulsions, etc., with or without subsequentsolvent removal, for example, by evaporation or extraction.Emulsification techniques are versatile and may be useful for preparingcore particles comprising pharmaceutical agents having a relatively lowaqueous solubility as well as pharmaceutical agents having a relativelyhigh aqueous solubility.

In some embodiments, the core particles described herein may be producedby emulsification in the presence of one or more surface-alteringagents. In some such embodiments, the stabilizer may act as asurface-altering agent, forming a coating on the particle (i.e., theemulsification and coating steps may be performed substantiallysimultaneously).

In some embodiments, a method of forming a core particle byemulsification involves choosing a stabilizer that is suitable for bothemulsification and for forming a coating on the particle and renderingthe particle mucus penetrating. For example, as described in more detailbelow, it has been demonstrated that 200-500 nm nanoparticles of a modelpolymer PLA produced by emulsification in the presence of certain PVApolymers resulted in particles that can penetrate physiological mucussamples at the same rate as well-established PEGylated polymeric MPP.Interestingly, it was observed that only a subset of PVA polymers testedfit the criteria of being suitable for both emulsification and forforming a coating on the particle that renders the particle mucuspenetrating, as described in more detail below.

In other embodiments, the particles are first formed using anemulsification technique, following by coating of the particles with asurface-altering agent.

Any suitable solvent and solvent combinations can be used foremulsification. Some examples of solvents which can serve as oil phaseare organic solvents such chloroform, dichloromethane, ethyl acetate,ethyl ether, petroleum ether (hexane, heptane), and oils such as peanutoil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oilsoybean oil, and silicone oil. Some examples of solvents which can serveas water phase are water and aqueous buffers. Other solvents are alsopossible.

The core and/or coated particles may also be formed by a precipitationprocess or technique (precipitation). Precipitation techniques (e.g.,microprecipitation, nanoprecipitation, crystallization, and controlledcrystallization) may involve forming a first solution comprising thematerial that is to form the core (e.g., a pharmaceutical agent) and afirst solvent, wherein the material has a relatively high solubility inthe first solvent. The first solution may be added to a second solutioncomprising a second solvent that is an anti-solvent, in which thematerial has a relatively low solubility, thereby forming a plurality ofparticles comprising the material. In certain embodiments, the secondsolvent is miscible with the first solvent. In some embodiments, one ormore surface-altering agents and/or surfactants may be present in thefirst and/or second solutions. A coating may be formed during theprocess of precipitating the core (e.g., the coating of the particlesmay be formed substantially simultaneously when the precipitation isperformed) to form the coated particles of the invention.

In other embodiments, the core of the particles of the invention isfirst formed using a precipitation technique, following by coating ofthe core with a surface-altering agent to form the coated particles ofthe invention.

In some embodiments, a precipitation technique may be used to formpolymeric core of the particles of the invention with or without apharmaceutical agent. Generally, a precipitation technique involvesdissolving a polymer that is to form the core in a first solvent, in thepresence or absence of a pharmaceutical agent, to form a solution. Thesolution is then added to a second solvent that is an anti-solvent andis miscible with the first solvent, in the presence or absence of one ormore excipients, to form the core of the particles. In some embodiments,precipitation is useful for preparing a polymeric core comprising one ormore pharmaceutical agents having a relatively low aqueous solubility.

The precipitation described herein involves the use of a first solvent.Examples of suitable first solvents for precipitation include organicsolvents (e.g., acetone, acetonitrile, dimethylformamide,dimethylsulfoxide, N-methyl-2-pyrrolidone, 2-pyrrolidone, andtetrahydrofuran) and inorganic solvents.

The precipitation described herein also involves the use of a secondsolvent. In certain embodiments, the second solvent suitable forprecipitation is an anti-solvent. Examples of second solvents suitablefor precipitation include the solvents described herein that may be usedfor milling. In some embodiments, the second solvents suitable forprecipitation is water, an aqueous solution (e.g., a buffered solution),an alcohol (e.g., methanol, ethanol, propanol, or butanol), or a mixturethereof, optionally including one or more other components, such aspharmaceutical excipients, polymers, and pharmaceutical agents.

Surface-altering agents for the emulsification and precipitationdescribed herein may be polymers or surfactants, including thesurface-altering agents described herein that may be used for milling.

Examples of polymers suitable for forming all or part of the core of theparticles of the invention by the emulsification or precipitation mayinclude polyamines, polyethers, polyamides, polyesters, polycarbamates,polyureas, polycarbonates, polystyrenes, polyimides, polysulfones,polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines,polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles,polyarylates, polypeptides, polynucleotides, and polysaccharides.Non-limiting examples of specific polymers include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),poly(ethylene glycol), poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) (jointlyreferred to herein as “polyacrylic acids”), and copolymers and mixturesthereof, polydioxanone and its copolymers, polyhydroxyalkanoates,polypropylene fumarate), polyoxymethylene, poloxamers,poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone, bovine serum albumin, human serum albumin,collagen, DNA, RNA, carboxymethyl cellulose, chitosan, dextran.

Polymers suitable for forming all or portions of a core and/orsurface-altering agent may also include a poly(ethylene glycol)-vitaminE conjugate (hereinafter, “PEG-VitE conjugate”). The particles,compositions, and/or formulations including a PEG-VitE conjugate, andmethods of making and using the particles, compositions, and/orformulations, are provided in more detail in international PCTapplication publication WO2012/061703, which is incorporated herein byreference in its entirety for all purposes. In some cases, the molecularweight of the PEG portion of the PEG-VitE conjugate is greater thanabout 2 kDa. The molecular weight of the PEG portion of the PEG-VitEconjugate may be selected so as to aid in the formation and/or transportof the particle across a mucosal barrier as described herein. In someembodiments, use of a PEG-VitE conjugate with a PEG portion having amolecular weight greater than about 2 kDa may allow for greaterpenetration of the particles through a mucosal barrier as compared touse of a PEG-VitE conjugate with a PEG portion having a molecular weightless than about 2 kDa. Additionally, in certain embodiments a highermolecular weight PEG portion may facilitate drug encapsulation. Thecombined ability to act as a surfactant and to reduce mucoadhesionprovides important benefits as compared to other commonly usedsurfactants for drug encapsulation. In some cases, the molecular weightof the PEG portion of the PEG-VitE conjugate is between about 2 kDa andabout 8 kDa, or between about 3 kDa and about 7 kDa, or between about 4kDa and about 6 kDa, or between about 4.5 kDa and about 6.5 kDa, orabout 5 kDa.

In some embodiments, a precipitation technique may be used to formparticles comprised predominantly of a pharmaceutical agent (e.g., acrystalline form of Compound 3). In certain embodiments, the particlesof the invention formed by the precipitation technique comprisepredominantly a crystalline form of Compound 3 that is a nanocrystal.Generally, such a precipitation technique involves dissolving Compound 3that is to form the core in a first solvent, which is then added to asecond solvent that is an anti-solvent, in which the crystalline form ofCompound 3 has a relatively low solubility, in the presence or absenceof one or more pharmaceutical excipients, to form the core or uncoatedparticle. In some embodiments, this technique may be useful forpreparing, for example, particles of pharmaceutical agents that areslightly soluble (1-10 mg/mL), very slightly soluble (0.1-1 mg/mL) orpractically insoluble (<0.1 mg/mL) in aqueous solutions (e.g., agentshaving a relatively low aqueous solubility).

A pharmaceutical agent described herein (e.g., a crystalline form ofCompound 3) may have a suitable solubility in the first and secondsolvents suitable for precipitation, such as a solubility in one or moreranges described herein for aqueous solubility or for solubility in acoating solution. A pharmaceutical agent having a relatively highsolubility in the first solvent (e.g., an organic solvent) may bepreferred. In certain embodiments, the pharmaceutical agentsubstantially or completely dissolves in the first solvent. Apharmaceutical agent having a relatively low solubility in the secondsolvent (e.g., water or a coating solution) may also be preferred. Incertain embodiments, the solubility of the pharmaceutical agent in amixture of the first and second solvents is lower than the solubility ofthe pharmaceutical agent in the first solvent. The relatively highsolubility and relatively low solubility are as described herein.

Another exemplary method of forming the core and/or coated particle is afreeze-drying process or technique known in the art. See, e.g., U.S.Patent Publication No. 2013/0316006. In this technique, Compound 3 maybe dissolved in an aqueous solution, optionally containing asurface-altering agent. The solution may be immediately flash frozen andfreeze dried. Dry powder can be reconstituted in a suitable solvent(e.g., an aqueous solution such as water) at a desired concentration.

If the surface-altering agent is present in the solvent prior to freezedrying, it may be present at any suitable concentration, such as aconcentration of at least about 0.001% (w/v), at least about 0.005%(w/v), at least about 0.01% (w/v), at least about 0.05% (w/v), at leastabout 0.1% (w/v), at least about 0.5% (w/v), at least about 1% (w/v), orat least about 5% (w/v) in the aqueous solution. In some instances, thesurface-altering agent is present in the solvent at a concentration ofless than or equal to about 5% (w/v), less than or equal to about 1%(w/v), less than or equal to about 0.5% (w/v), less than or equal toabout 0.1% (w/v), less than or equal to about 0.05% (w/v), less than orequal to about 0.01% (w/v), or less than or equal to about 0.005% (w/v).Combinations of the above-referenced ranges are also possible (e.g., aconcentration of at least about 0.01% (w/v) and less than or equal toabout 1% (w/v). Other ranges are also possible.

The concentration of surface-altering agent present in the solvent maybe above or below the critical micelle concentration (CMC) of thesurface-altering agent, depending on the particular surface-alteringagent used. In other embodiments, stable particles can be formed byadding excess counter-ion to a solution containing a pharmaceuticalagent. The precipitate can then be washed by various methods such ascentrifugation. The resultant slurry may be sonicated. One or moresurface-altering agents may be added to stabilize the resultantparticles.

Other methods of forming core particles are also possible. For example,additional techniques of forming the core and/or coated particlesinclude coacervation-phase separation, melt dispersion, interfacialdeposition, in situ polymerization, self-assembly of macromolecules(e.g., formation of polyelectrolyte complexes orpolyelectrolyte-surfactant complexes), spray-drying andspray-congealing, electro-spray, air suspension coating, pan and spraycoating, freeze-drying, air drying, vacuum drying, fluidized-bed drying,precipitation (e.g., nanoprecipitation, microprecipitation), criticalfluid extraction, and lithographic approaches (e.g., soft lithography,step and flash imprint lithography, interference lithography, andphotolithography). Combinations of the methods described herein are alsopossible. In some embodiments, a core of a pharmaceutical agent is firstformed by precipitation, and then the size of the core is reduced by amilling process, optionally a coating is form on the core by the millingprocess.

Following the formation of the core of the particles including apharmaceutical agent, the core may be optionally exposed to a solutioncomprising a (second) surface-altering agent that may associate withand/or coat the core. In embodiments in which the pharmaceutical agentalready includes a coating of a first surface-altering agent, all orpart of the first surface-altering agent may be exchanged with a secondsurface-altering agent. In some embodiments, the second surface-alteringagent renders the particle mucus penetrating more than the firstsurface-altering agent does. In some embodiments, a particle having acoating including multiple surface-altering agents is formed (e.g., in asingle layer or in multiple layers). In some embodiments, a particlehaving multiple coatings (e.g., each coating optionally comprisingdifferent surface-altering agents) may be formed. In some embodiments,the coating is in the form of a monolayer of a surface-altering agent.Other configurations are also possible.

In any of the methods described herein, a coating comprising asurface-altering agent may be formed on a core of the particles of theinvention by incubating the core in a solution including thesurface-altering agent for a period of at least about 1 minute, at leastabout 3 minutes, at least about 10 minutes, at least about 20 minutes,at least about 30 minutes, at least about 60 minutes, or more. In somecases, incubation may take place for a period of less than about 10hours, less than about 3 hours, or less than about 60 minutes.Combinations of the above referenced ranges are also possible (e.g., anincubation period of less than 60 minutes and at least about 1 minute).

Methods of Treatment and Uses

A range of diseases may result when the body of a subject loses controlover angiogenesis, i.e., new blood vessels grow abnormally (i.e.,excessively or insufficiently) or grow as a result of a tumor. Excessiveangiogenesis is often observed in subjects with diseases such asproliferative diseases (e.g., cancers, benign neoplasms, inflammatorydiseases, autoimmune diseases) and ocular diseases, especially withcancer, diabetic retinopathy, macular degeneration, rheumatoidarthritis, and psoriasis. In these diseases, new blood vessels feedabnormal tissues and/or destroy normal tissues. Excessive angiogenesismay occur when there are abnormal amounts of angiogenic growth factorspresent, overwhelming the effects of natural angiogenesis inhibitors.Therefore, inhibiting new blood vessel growth may be useful to treatdiseases associated with excessive angiogenesis. Insufficientangiogenesis is typically observed in subjects with a disease such ascoronary artery disease, stroke, or chronic wounds. In these diseases,blood vessel growth is inadequate, and circulation is not properlyrestored, which may lead to tissue death.

VEGFs have been found to play a major role in angiogenesis, for example,by increasing the number of capillaries in a given network. In vitrostudies have demonstrated that bovine capillary endothelial cellsproliferated and showed signs of tube structures upon stimulation withVEGF. Upregulation of VEGF is a major component of the physiologicalresponse to exercise and its role in angiogenesis is suspected to be apossible treatment in vascular injuries. In vitro studies have showedthat VEGFs are a potent stimulator of angiogenesis because, among otherthings, in the presence of this growth factor, plated endothelial cellswill proliferate and migrate, eventually forming tube structuresresembling capillaries. VEGFs may cause a massive signaling cascade inendothelial cells. Binding to VEGF receptor-2 starts a tyrosine kinasesignaling cascade that stimulates the production of factors thatvariously stimulate vessel permeability, proliferation/survival,migration, and finally differentiation into mature blood vessels.Mechanically, VEGF is upregulated with muscle contractions as a resultof increased blood flow to affected areas. The increased flow alsocauses a large increase in the mRNA production of VEGF receptors 1 and2. The increase in receptor production indicates that musclecontractions could cause upregulation of the signaling cascade relatingto angiogenesis.

In one aspect, the present invention provides methods of treating and/orpreventing a disease associated with abnormal angiogenesis, whichcomprise administering an effective amount of Compound 3 to a subject inneed thereof. In certain embodiments, the disease associated withabnormal angiogenesis is treated and/or prevented by the inventivemethods. In certain embodiments, the disease being treated and/orprevented by the inventive methods is associated with excessive and/orpathological angiogenesis.

In another aspect, the present invention provides methods of treatingand/or preventing a disease associated with aberrant signaling of agrowth factor in a subject in need thereof. In certain embodiments, thedisease associated with aberrant signaling of a growth factor is treatedand/or prevented by the inventive methods. In certain embodiments, thedisease is associated with excessive signaling of the growth factor. Incertain embodiments, the disease being treated and/or prevented by theinventive methods is associated with aberrant signaling of VEGF. Incertain embodiments, the disease is associated with excessive oraberrant signaling of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-F, and/orplacental growth factor (PGF). In certain embodiments, the diseaseassociated with aberrant signaling of VEGF is treated and/or preventedby the inventive methods.

As used herein, the term “growth factor-associated disease” means anydisease where growth factors are known to play a role. Accordingly, insome embodiments, the present disclosure relates to treating diseases inwhich growth factors are known to play a role. Such diseases includeproliferative diseases, eye diseases, dermatological diseases,inflammation diseases, and metabolic diseases.

In some embodiments, the present disclosure provides methods of treatinga disease comprising contacting a biological sample with an effectiveamount of Compound 3. In certain embodiments, the biological sampleincludes a cell or tissue. In some embodiments, the methods compriseinhibiting growth factor signaling in a cell, tissue, or subject. Insome embodiments, the biological sample is an ocular tissue. In certainembodiments, the method is an in vitro method. In certain embodiments,the method is an in vivo method. It will be understood by one ofordinary skill in the art that levels of inhibition are not necessary tobe 100%. The levels of inhibition can be at least 10% inhibition, about10% to about 25% inhibition, about 25% to about 50% inhibition, about50% to about 75% inhibition, at least 50% inhibition, at least 75%inhibition, about 80% inhibition, about 90% inhibition, or greater than90% inhibition.

In certain embodiments, the disease being treated and/or prevented bythe inventive methods is a proliferative disease. All types ofproliferative diseases described herein may be treated and/or preventedby the inventive methods. In certain embodiments, the proliferativedisease is treated and/or prevented by the inventive methods. In certainembodiments, the disease being treated and/or prevented by the inventivemethods is cancer. All types of cancer described herein may be treatedand/or prevented by the inventive methods. In certain embodiments, thecancer is an ocular cancer. In certain embodiments, the ocular cancer isretinoblastoma, medulloepithelioma, uveal melanoma, ciliary bodymelanoma, or primary intraocular lymphoma. In certain embodiments, thecancer is treated and/or prevented by the inventive methods. In certainembodiments, the disease being treated and/or prevented by the inventivemethods is a benign neoplasm. All types of benign neoplasm describedherein may be treated and/or prevented by the inventive methods. Incertain embodiments, the benign neoplasm is an ocular benign neoplasm.In certain embodiments, the benign neoplasm is orbital dermoid cysts. Incertain embodiments, the benign neoplasm is treated and/or prevented bythe inventive methods.

In certain embodiments, the disease being treated and/or prevented bythe inventive methods is an inflammatory disease. All types ofinflammatory diseases described herein may be treated and/or preventedby the inventive methods. In certain embodiments, the inflammatorydisease is an ocular inflammatory disease. In certain embodiments, theocular inflammatory disease is post-surgical inflammation. In certainembodiments, the inflammatory disease is treated and/or prevented by theinventive methods. In certain embodiments, the disease being treatedand/or prevented by the inventive methods is an autoimmune disease. Alltypes of autoimmune diseases described herein may be treated and/orprevented by the inventive methods. In certain embodiments, theautoimmune disease is rheumatoid arthritis. In certain embodiments, theautoimmune disease is treated and/or prevented by the inventive methods.In certain embodiments, the disease being treated and/or prevented bythe inventive methods is diabetes. In certain embodiments, the diseaseis type 1 diabetes. In certain embodiments, the disease is type 2diabetes. In certain embodiments, the disease is gestational diabetes.In certain embodiments, the diabetes is treated and/or prevented by theinventive methods.

The disease being treated and/or prevented by the inventive methods maybe an ocular disease. In some embodiments, the ocular disease beingtreated and/or prevented by the inventive methods is an anterior oculardisease that occurs at the anterior portion or “front” of the eye of asubject. The anterior portion of the eye includes the cornea, iris,conjunctiva, tear film, corneal epithelium, anterior chamber, lens,ciliary body, ciliary zonule, posterior chamber, retina, macula, sclera,an optic nerve, choroid, and vitreous chamber. In certain embodiments,the anterior ocular disease being treated and/or prevented by theinventive methods is allergy, post-surgical inflammation, uveitis, aninfection (e.g., a viral, bacterial, or fungal infection), aphakia,pseudophakia, astigmatism, blepharospasm, cataract, a conjunctivaldisease, conjunctivitis, a corneal disease, corneal edema, blepharitis,meibomian gland disease, corneal transplant surgery, corneal ulcer, dryeye (e.g., dry eye syndrome), an eyelid disease, a lacrimal apparatusdisease, lacrimal duct obstruction, laser induced exudation, myopia,presbyopia, pterygium, pupil disorders, corneal neovascularization, arefractive disorder, strabismus, or glaucoma. In some embodiments, theocular disease being treated and/or prevented by the inventive methodsis a posterior ocular disease that occurs at the posterior portion or“back” of the eye. The posterior portion of the eye includes thechoroid, sclera, vitreous humor, vitreous chamber, retina, macula, opticnerve, and blood vessels and nerves which vascularize or innervate aposterior ocular region or site. In certain embodiments, the posteriorocular disease being treated and/or prevented by the inventive methodsis intraocular melanoma, acute macular neuroretinopathy, an exudativeeye disease, Behcet's disease, exudative retinopathy, macular edema,retinopathy of prematurity, an epiretmal membrane disorder, choroidalneovascularization, uveitis, diabetic uveitis, histoplasmosis, aninfection (e.g., a viral, bacterial, or fungal infection), maculardegeneration (e.g., acute macular degeneration and age-related maculardegeneration (AMD, such as non-exudative age-related maculardegeneration and exudative age-related macular degeneration)), edema(e.g., macular edema, such as cystoid macular edema (CME) and diabeticmacular edema (DME)), multifocal choroiditis, ocular trauma whichaffects a posterior ocular site or location, ocular cancer, a retinaldisorder (e.g., central retinal vein occlusion), diabetic retinopathy(e.g., proliferative diabetic retinopathy and non-proliferative diabeticretinopathy), proliferative vitreoretinopathy (PVR), retinal arterialocclusive disease, retinal detachment, uveitic retinal disease,sympathetic opthalmia, Vogt Koyanagi-Harada (VKH) syndrome, uvealdiffusion, a posterior ocular condition caused by or influenced by anocular laser treatment, a posterior ocular condition caused by orinfluenced by a photodynamic therapy, photocoagulation, radiationretinopathy, an epiretinal membrane disorder, branch retinal veinocclusion, anterior ischemic optic neuropathy, non-retinopathy diabeticretinal dysfunction, retinitis pigmentosa, retinoblastoma, or glaucoma.In certain embodiments, the ocular disease being prevented and/ortreated by the inventive methods is macular degeneration. In certainembodiments, the ocular disease is age-related macular degeneration(AMD). In certain embodiments, the ocular disease is glaucoma. Incertain embodiments, the ocular disease is diabetic retinopathy. Incertain embodiments, the ocular disease is retinoblastoma. In certainembodiments, the ocular disease is edema. In certain embodiments, theocular disease is cystoid macular edema (CME). In certain embodiments,the ocular disease is diabetic macular edema (DME). In certainembodiments, the ocular disease is an ocular inflammatory disease. Incertain embodiments, the ocular disease is post-surgical inflammation.In certain embodiments, the ocular disease is uveitis (e.g., anterioruveitis, intermediate uveitis, and posterior uveitis). In certainembodiments, the ocular disease is blepharitis. In certain embodiments,the ocular disease is panuveitis. In certain embodiments, the oculardisease is scleritis. In certain embodiments, the ocular disease is dryeye. In certain embodiments, the ocular disease is Sjögren's syndrome.In certain embodiments, the ocular disease is an eye surgery. In certainembodiments, the ocular disease is treated and/or prevented by theinventive methods.

In certain embodiments, the compounds, particles, compositions, and/orformulations described herein are packaged as a ready to use shelfstable suspension. Eye drop formulations are traditionally liquidformulations (solutions or suspensions) which can be packaged in dropperbottles (which dispense a standard drop volume of liquid) or inindividual use droppers (typically used for preservative free drops,used once and disposed). These formulations are ready to use and can beself-administered. In some cases the bottle should be shaken before useto ensure homogeneity of the formulation, but no other preparation maybe necessary. This may be the simplest and most convenient method ofocular delivery. The compositions and/or formulations described hereincan be packaged in the same way as traditional eye drop formulations.

Another aspect of the present invention relates to methods of inhibitingthe aberrant signaling of a growth factor (e.g., VEGF) signaling pathwayin a subject or cell. In certain embodiments, the aberrant signaling ofthe growth factor is inhibited by the inventive methods.

In another aspect, the present invention provides methods of inhibitingthe abnormal or pathological angiogenesis in a subject in need thereof.In certain embodiments, the abnormal or pathological angiogenesis isinhibited by the inventive methods.

In certain embodiments, the subject described herein is a human. Incertain embodiments, the subject is an animal. The animal may be ofeither sex and may be at any stage of development. In certainembodiments, the subject is a fish. In certain embodiments, the subjectis a mammal. In certain embodiments, the subject is a domesticatedanimal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certainembodiments, the subject is a companion animal such as a dog or cat. Incertain embodiments, the subject is a livestock animal such as a cow,pig, horse, sheep, or goat. In certain embodiments, the subject is a zooanimal. In another embodiment, the subject is a research animal such asa rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certainembodiments, the animal is a genetically engineered animal. In certainembodiments, the animal is a transgenic animal.

In some embodiments, the crystalline forms described herein are usefulfor treating a cancer including, but not limited to, acoustic neuroma,adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett'sadenocarcinoma), Ewing sarcoma, eye cancer (e.g., intraocular melanoma,retinoblastoma), familiar hypereosinophilia, gall bladder cancer,gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromaltumor (GIST), head and neck cancer (e.g., head and neck squamous cellcarcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC),throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngealcancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemiasuch as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL),acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLUSLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., “Waldenström's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, smallcell lung cancer (SCLC), non-small cell lung cancer (NSCLC),adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM),a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronicmyelocytic leukemia (CML), chronic neutrophilic leukemia (CNL),hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g.,neurofibromatosis (NF) type 1 or type 2, schwannomatosis),neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrinetumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g.,cystadenocarcinoma, ovarian embryonal carcinoma, ovarianadenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g.,pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm(IPMN), islet cell tumors), penile cancer (e.g., Paget's disease of thepenis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT),prostate cancer (e.g., prostate adenocarcinoma), rectal cancer,rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamouscell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cellcarcinoma (BCC)), small bowel cancer (e.g., appendix cancer), softtissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma,malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma,fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat glandcarcinoma, synovioma, testicular cancer (e.g., seminoma, testicularembryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of thethyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer),urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's diseaseof the vulva).

In certain embodiments, the cell described herein is in vivo. In certainembodiments, the cell is in vitro. In certain embodiments, the cell isex vitro.

In certain embodiments, the methods of the invention includeadministering to a subject in need thereof an effective amount ofCompound 3, particles, or pharmaceutical composition of the invention.In certain embodiments, the methods of the invention include contactinga cell with an effective amount of a compound, particles, orpharmaceutical composition of the invention.

In certain embodiments, the inventive methods are in vivo methods. Incertain embodiments, the inventive methods are in vitro methods. Incertain embodiments, the inventive methods are ex vitro methods.

In another aspect, the present invention provides the crystalline formsof Compound 3, particles, and pharmaceutical compositions of theinvention for use in the treatment and/or prevention of a diseasedescribed herein in a subject in need thereof.

In yet another aspect, the present invention provides the crystallineforms of Compound 3, particles, and pharmaceutical compositions of theinvention for use in the inhibition of abnormal angiogenesis in asubject in need thereof.

In still another aspect, the present invention provides the crystallineforms of Compound 3, particles, and pharmaceutical compositions of theinvention for use in the inhibition of aberrant signaling of a growthfactor in a subject or cell in need thereof.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Example 1 Synthesis of Compound 3, Method A Compound 1:4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-7-(benzyloxy)-6-methoxyquinazoline

4-Fluoro-2-methyl-1H-indol-5-ol (0.53 g, 3.2 mmol) was dissolved inN,N-dimethylformamide (25 mL). The suspension was purged with nitrogenand potassium carbonate (0.92 g, 6.7 mmol) was added.7-(Benzyloxy)-4-chloro-6-methoxyquinazoline (1.0 g, 3.3 mmol) was addedand the suspension was purged with nitrogen again. The suspension washeated overnight at 85° C. in an oil bath. The solvent was evaporated.The residue was treated with water (100 mL) and sonicated. The solid wasfiltered off, washed with water and hexanes, and dried in high vacuumovernight leaving Compound 1 as a gray solid (1.4 g, 100%). m/z: 430(M+H, 100%) (positive ionization mode).

Compound 2:4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinazolin-7-ol

4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-7-(benzyloxy)-6-methoxyquinazoline(Compound 1, 0.46 g, 1.1 mmol) was dissolved in N,N-dimethylformamide(10 mL). Palladium hydroxide catalyst (250 mg, 10% on carbon) was added,followed by ammonium formate (0.67 g, 10.6 mmol). The reaction solutionwas stirred for 2 hours at room temperature. The catalyst was filteredthrough a CELITE pad, then the solution was evaporated, then dried inhigh vacuum overnight to generate Compound 2 as a brown solid (0.36 g,100%) m/z: 340 (M+H, 100%) (positive ionization mode).

Compound 3:7-(3-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinazolin-7-yloxy)propyl)-2-oxa-7-azaspiro[3.5]nonane

4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinazolin-7-ol(Compound 2, 0.36 g, 1.1 mmol) was dissolved in N,N-dimethylformamide(10 mL). Potassium carbonate (0.90 g, 6.5 mmol) was added followed by1-bromo-3-chloropropane (0.34 g, 2.2 mmol). The suspension was heated at45° C. for 2 hours. The solvent was evaporated and the residue wassuspended in dichloromethane (20 mL). The suspension was applied on apad of silica gel. The impurities were eluted with dichloromethane andthe compound was eluted with ethyl acetate (Rf=0.7 in ethyl acetate).The solvent was evaporated and the residue was dried in high vacuumleaving a yellow foam (0.35 g, 80%) m/z: 416 (M+H, 100%) (positiveionization mode), which was dissolved in N,N-dimethylformamide (5 mL).Potassium bromide (0.12 g, 1.0 mmol) was added followed by potassiumcarbonate (0.90 g, 7.8 mmol) and 2-oxa-7-azaspiro[3.5]nonane oxalate(0.35 g, 1.9 mmol). The suspension was heated at 85° C. for 4 hours. Thesolvent was evaporated and the residue was suspended in aqueous sodiumbicarbonate (50 mL) and sonicated. The precipitate was filtered off.Drying in high vacuum gave a brown solid (0.34 g). Purification byreverse phase HPLC provided Compound 3 as an off-white solid (20 mg).m/z: 507 (M+H, 100%) (positive ionization mode). 1H NMR: (Chloroform-d):8.60 (s, 1H), 8.10 (s, 1H), 7.65 (s, 1H); 7.35 (s, 1H), 7.10 (d, J=9.0Hz 1H), 7.00 (dd, J=8.0; J=9.0 Hz, 1H), 6.35 (s, 1H), 4.45 (s, 4H), 4.35(t, J=7.0 Hz, 2H), 4.15 (s, 3H), 2.55 (t, J=7.0 Hz, 2H), 2.46 (s, 3H),2.40-2.35 (m, 4H), 2.15-2.10 (m, 2H), 1.90-1.85 (m, 4H).

Example 2 Synthesis of Compound 3, Method B

To a solution of 2-amino-4-(3-chloropropoxy)-5-methoxybenzoic acidmethyl ester (48 g, 175 mmol) in methanol (150 mL) was added methylorthoformate (46.4 g, 438 mmol), ammonium acetate (33.7 g, 438 mmol).The reaction mixture was stirred at reflux for 5 hours. Water (200 mL)was added to the reaction mixture to precipitate product, which wascollected by filtration, washed with water (200 mL) and methanol (50mL), then dried under reduced pressure to give 44 g (93.4%) of Compound4 as a white solid.

A mixture of Compound 4 (75 g, 279 mmol) and POCl₃ (100 mL) in toluene(500 mL) was stirred at reflux until the solution became clear. Thesolution was concentrated under reduced pressure and the residue waspoured into ice water. After filtration, the solid was washed with water(500 mL×2) and dried to give 65 g (81.2%) of Compound 5 as a yellowsolid.

The mixture of Compound 5 (11 g, 38.33 mmol) and4-fluoro-5-hydroxy-2-methylindole (9.49 g 57.50 mmol), Cs₂CO₃ (25 g,76.66 mmol) in tetrahydrofuran (200 mL) was stirred at 50° C. overnight.The reaction mixture was extracted with ethyl acetate (200 mL×2) and theorganic layers were combined and washed with water (200 mL) and brine(200 mL) successively. The organic layer was dried with sodium sulfate,then concentrated to dryness. The residue was purified by flashchromatography (petroleum ether:ethyl acetate=10:1 to 2:1) to give 13 g(81.5%) of Compound 6 as a brown solid.

To the solution of Compound 6 (11 g, 26.44 mmol) inN,N-dimethylformamide (100 mL) was added 2-oxa-7-azaspiro[3.5]nonane(8.73 g, 68.75 mmol), tetrabutylammonium iodide (9.76 g, 26.44 mmol) anddiisopropylethylamine (10.23 g, 79.33 mmol). The solution was heated to60° C. overnight, then diluted with ethyl acetate (200 mL). The mixturewas washed with brine (100 mL×5), dried over sodium sulfate, thensolvent was evaporated under reduced pressure to give the crude productas a black solid. The crude product was purified by flash chromatography(dichloromethane:methanol=50:1 to 10:1) to give 6.5 g (48.5%) ofCompound 3 as a light yellow solid. Analysis by XRPD showed that theisolated compound was amorphous.

Example 3 Compound 3 Formulated as Mucus Penetrating Particles (MPP)

Compound 3 was formulated as mucus penetrating particles (MPP).Specifically, Compound 3 from Example 2 was milled in the presence ofPLURONIC F127 (F127) to determine whether F127 1) aids particle sizereduction to several hundreds of nanometers and 2) physically(non-covalently) coats the surface of generated nanoparticles with amucoinert coating that would minimize particle interactions with mucusconstituents and prevent mucus adhesion.

A milling procedure was employed in which an aqueous dispersioncontaining coarse drug particles and PLURONIC F127 (F127) was milledwith grinding medium until particle size was reduced to approximately270 nm (z-averaged) as measured by dynamic light scattering. Theseparticles were found to have a polydispersity index (a measure of thewidth of the particle size distribution) of 0.142. In this examplesuspensions were buffered using DPBS (Dulbecco's Phosphate-BufferedSaline) which yields a suspension that is both isotonic and has aphysiologically relevant pH.

In order to determine whether the generated particles have reducedinteractions with mucins and are therefore able to move within mucuswithout becoming trapped, particles were incubated with humancervicovaginal mucus (CVM) and observed via dark field microscopy. 1 μLor less of the nanoparticle suspension was added to 20 μL of CVM.Observations were made in a minimum of three distinct and randomlyselected areas of the CVM sample. Control particles with known behaviorwere used to qualify the CVM sample as appropriate for the assay.Mobility in mucus was observed and therefore the nanoparticles weredeemed to be effective MPP.

Example 4 Crystalline Forms of Compound 3

The crystalline forms of Compound 3 were prepared, and then analyzed byXRPD, Differential Scanning Calorimetry (DSC) analysis andThermogravimetric Analysis (TGA).

For XRPD, patterns were obtained using a Rigaku MiniFlex 600 benchtopx-ray diffractometer equipped with a Cu X-ray tube (Cu/Kα=1.54059 Å), asix-position sample changer and a D/teX Ultra detector.

Sample Preparation, Procedure A

As described below for the preparation of Crystalline Form A by milling,particles were isolated from bulk formulation by centrifugation at55,000 rpm over 15 minutes and deposited thinly and evenly onto a flatzero background XRPD sample holder (Rigaku 906165 Flush, Si510). Thesample was allowed to dry under gentle air stream, usually for up to 3minutes, until it was visually dry.

Sample Preparation, Procedure B

As described below for the preparation of neat Crystalline Form B bycrystallization, milligram amounts of solid sample were firmly packed inthe 5-mm×0.2-mm depression of a zero background sample holder (Rigaku906166 5 mm×0.2 mm Well, Si510).

XRPD patterns were acquired from 3-40° two theta at 0.02° step size and5°/min scan speed using the following instrument settings: 40 kV-15 mAX-ray generator, 2.5° Soller Slit, 10 mm HIS, 0.625° Divergence Slit, 8mm Scatter Slit with Kβ filter, and an open Receiving Slit. Diffractionpatterns were viewed and analyzed using PDXL analysis software providedby the instrument manufacturer. Using the sample preparation proceduresdescribed above, a reference standard silicon powder (NIST StandardReference Material 640d) generated a peak at 28.44° and 28.38° two thetausing Procedure A and Procedure B, respectively.

For DSC, about 2 mg of sample was weighed into a standard aluminumsample pan. The sample pan was loaded into the apparatus (Q1000Differential Scanning Calorimeter, TA Instruments), which was equippedwith an autosampler. A thermogram was obtained by individually heatingthe sample at a rate of 10° C./min from room temperature toapproximately 250-300° C. using an empty standard aluminum pan as areference. Dry nitrogen was used as a sample purge gas and was set toflow at 50 mL/min. Thermal transitions were viewed and analyzed usingthe analysis software provided with the instrument.

For TGA, about 6 mg of the sample was transferred into an aluminumsample pan. The pan was placed in the loading platform and was thenautomatically loaded into the apparatus (Q500 ThermogravimetricAnalyzer, TA Instruments) using the control software. Thermograms wereobtained by individually heating the sample at 10° C./min from roomtemperature to 300° C. under flowing dry nitrogen, with a sample purgeflow rate of 25 mL/min and a balance purge flow rate of 10 mL/min.Thermal transitions (e.g., weight changes) were viewed and analyzedusing the analysis software provided with the instrument.

Preparation of Mucus-Penetrating Particles Comprising of CrystallineForm A

In accordance to Example 3, mucus-penetrating particles comprising ofCrystalline Form A was prepared by wet milling amorphous Compound 3produced in Example 2. A slurry containing 5% amorphous Compound 3 and5% F127 in PBS (0.0067 M PO₄ ³⁻), pH 7.1 was added to an equal bulkvolume of 1-mm ceria-stabilized zirconium oxide beads in a glass vial(e.g., 2 mL of slurry per 2 mL of beads). A magnetic stir bar was usedto agitate the beads, stirring at approximately 500 rpm. The sample wasmilled for 2 days. Nanoparticles which were approximately 200 nm(z-averaged) in diameter, as measured by dynamic light scattering (DLS),were generated. During the milling process, Compound 3 converted fromamorphous to crystalline Form A as confirmed by XRPD.

An XRPD analysis of the resulting crystalline Form A of Compound 3 wasperformed. The XRPD pattern of crystalline Form A is illustrated in FIG.1 and the reflections comprised in its XRPD pattern are listed in Table1.

TABLE 1 XRPD Peak Listing for Crystalline Form A of Compound 3 Position± 0.3 d-spacing ± 0.3 Relative Intensity No. [°2θ] [Å] [%] 1 6.11 14.4560.14 2 9.63 9.17 52.72 3 11.10 7.96 11.17 4 11.46 7.71 22.60 5 12.267.22 10.66 6 15.66 5.65 3.25 7 16.41 5.40 30.14 8 17.54 5.05 7.29 918.16 4.88 44.67 10 18.60 4.77 100 11 19.51 4.55 32.84 12 20.36 4.3652.26 13 21.12 4.20 12.65 14 22.31 3.98 7.49 15 23.01 3.86 24.04 1624.79 3.59 7.64 17 25.71 3.46 30.32 18 28.90 3.09 8.29 19 30.81 2.901.99 20 31.64 2.83 3.28Preparation of Neat Crystalline Form B

Crystalline Form B of Compound 3 was prepared by thermal crystallizationof the amorphous form of Compound 3 in a binary mixture of acetone andwater. Specifically, Compound 3 (80 mg) from Example 2 was added to an8-mL scintillation vial containing a 7×2 mm stir bar, followed byaddition of a hot mixture of 4:1 acetone:water (4 mL total). The vialwas heated on a hot plate while stirring to completely dissolve Compound3. Upon spontaneous cooling to ambient temperature, Form B crystallizedslowly from solution. After allowing crystallization to continueovernight, solvent was discarded and the solid crystals that remained inthe vial were collected and allowed to dry overnight under vacuum. Asdiscussed below, XRPD analysis generated unique reflections, indicatingthe formation of a new crystalline form.

An XRPD analysis of the resulting crystalline Form B of Compound 3 wasperformed. The XRPD pattern of crystalline Form B is illustrated in FIG.2 and the reflections comprised in its XRPD pattern are listed in Table2.

TABLE 2 XRPD Peak Listing for Crystalline Form B of Compound 3 Position± 0.3 d-spacing ± 0.3 Relative Intensity No. [°2θ] [Å] [%] 1 7.7 11.47 72 9.87 8.96 5 3 10.69 8.27 4 4 12.88 6.87 1 5 13.53 6.54 30 6 14.4 6.1412 7 14.97 5.91 5 8 15.45 5.73 12 9 16.42 5.39 3 10 17.27 5.13 42 1118.44 4.81 100 12 18.9 4.69 3 13 19.73 4.5 20 14 21.14 4.2 8 15 21.864.06 2 16 22.56 3.94 14 17 23.1 3.85 67 18 26.07 3.41 72 19 26.84 3.32 820 29.12 3.06 9

DSC and TGA were also conducted on crystalline Form B of Compound 3.FIG. 3 shows that the DSC thermogram measured from 25° C. to 250° C.,ramped at 10° C./min, was found to exhibit a broad dehydrationendothermic event at 117° C. followed by crystallization then themelting of the presumed anhydrous form at 188° C. FIG. 4 shows that theTGA thermogram measured from 25° C. to 300° C., ramped at 10° C./min,was found to exhibit a mass loss of 6% from 25° C. up to 120° C.Presumably, the mass loss corresponds to two water molecules(theoretical weight loss of dihydrate=6.6%), thus making crystallineform B a dihydrate.

Preparation of Mucus-Penetrating Particles Comprising of CrystallineForm B

In accordance to Example 3, mucus-penetrating particles comprising ofCrystalline Form B was prepared by wet-milling neat Crystalline Form B.A slurry containing 5% neat crystalline From B of Compound 3 and 5% F127in DPBS (Dulbecco's Phosphate Buffered Saline) was added to an equalbulk volume of 1-mm ceria-stabilized zirconium oxide beads in a glassvial (e.g. 0.5 mL of slurry for 0.5 mL of beads). A magnetic stir barwas used to agitate the beads, stirring at approximately 500 rpm. Thesample was milled for 2 days. Nanoparticles which were approximately 200nm in diameter (z-averaged), as measured by DLS, were generated. Aftermilling, XRPD analysis (not shown) confirmed that the crystal form wasunchanged, which indicates that crystalline Form B remained stableduring milling.

Stability of MPP Formulations Containing Crystalline Forms A and/or B

To characterize the stability of the resulting crystalline forms ofCompound 3, changes in XRPD profiles following long-term storage weredetermined. An MPP suspension comprising of crystalline Form A wasstored at room temperature for 7 weeks, while a second MPP suspensionalso comprising of crystalline Form A was stored at room temperature for7 weeks followed by an additional 1.5 weeks of agitation. Followingthese periods, XRPD analyses revealed that the compounds still possessedthe XRPD profile of crystalline Form A, indicating that the material isshelf stable in solution for at least 7 weeks. To further test thepotential for longer term storage, two additional MPP samples comprisingof Form A, which were formulated at pH 5.8 and pH 7.4 by incorporatingdifferent buffers in the milling slurry, were stored at room temperaturefor 8 months and then analyzed by XRPD. Again, the XRPD analysis (notshown) revealed that the crystals still possessed the XRPD profile ofcrystalline Form A, indicating that the materials are shelf stable assuspensions for at least 8 months.

To confirm long-term stability, an MPP suspension of crystalline Form Bwas stored for 7 weeks at room temperature and then was characterized byXRPD. The results of that analysis are shown in FIG. 5 , which providesthe XRPD pattern of the original crystalline Form B material in thebottom trace and the XRPD pattern of the material after seven weeks ofstorage in the top trace. The material remained as crystalline Form B,which demonstrates that crystalline Form B is physically stable duringstorage.

Seeding Crystalline Form B During Milling of Amorphous Compound 3

In the absence of crystalline material, the amorphous material fromExample 2 becomes crystalline Form A during milling, as described above.As such, the inventors wished to determine whether the presence ofcrystalline Form B during milling would seed the formation ofcrystalline Form B from amorphous Compound 3 during milling. A mixtureof crystalline Form B and amorphous material was milled as follows:milling media, specifically 1-mm ceria-stabilized zirconium oxide beads,was added to a glass scintillation vial. Separately, a milling slurrywas generated containing 2.5% crystalline Form B of Compound 3, 2.5%amorphous Compound 3, and 5% F127 in DPBS (Dulbecco's Phosphate BufferedSaline). The milling slurry was then added to the glass vial at an equalbulk volume to the beads (e.g. 0.5 mL of slurry for 0.5 mL of beads). Amagnetic stir bar was used to agitate the beads, stirring atapproximately 500 rpm. The sample was milled for 3 days. Nanoparticleswhich were approximately 150 nm (z-averaged) in diameter, as measured byDLS, were generated. After milling, XRPD analysis was performed, theresults of which are shown in FIG. 6 , which provides the XRPD patternof crystalline Form B of Compound 3 in the bottom trace and the XRPDpattern of the milling mixture of amorphous and crystalline Form B ofCompound 3 in the top trace. This analysis confirmed that, when somecrystalline Form B was present during the milling process, the amorphousmaterial converted to crystalline Form B as opposed to crystalline FormA during milling.

Competition Between Crystalline Forms A and B to Determine the MoreStable or Preferred MPP Form

In order to assign one form as more stable, or more preferred, under thecurrent formulation conditions, a competition experiment was performed.A suspension of nanoparticles of crystalline Form A was generated asdescribed via wet milling the amorphous material. A suspension ofnanoparticles of Form B was generated via wet milling of neat Form Bcrystals as described. The two suspensions were mixed in 1:1 ratio andincubated at room temperature. After 11 days, an XRPD analysis wasperformed, which estimated that the ratio of Forms A and B was unchangedin the mixture (not shown). A fraction of the mixture was then stirredusing a magnetic stir bar to provide an increased energy input in orderto accelerate the outcome of the competition experiment. After 5 weeksof stirring, the Compound 3 in the stirred formulation converted tocrystalline Form B whereas the crystals in the unstirred formulationremained a mixture, as shown in FIG. 7 . This result indicates thatcrystalline Form B is more stable under the current formulationconditions.

Example 5 Back of the Eye Drug Exposure from Topical Instillation of anMPP Comprising Crystalline Form A of Compound 3

A pharmacokinetic (PK) study of the crystalline Form A of Compound 3formulated as MPP in accordance with Example 4 was performed in order todemonstrate that topical instillation of MPP formulations of thesecompounds results in drug exposure at the back of the eye. The studydesign is shown in Table 3.

TABLE 3 Study design for PK evaluation of Compound 3, Form A MPP Numberof Terminal Animals Time Test (n/time Dose Frequency/ Points GroupArticle point) Volume Duration (hours) 1 3, Form A 3 35 μL BID/5 days0.5 MPP, 2.0% 2 3, Form A 3 35 μL BID/5 days 1 MPP, 2.0% 3 3, Form A 335 μL BID/5 days 2 MPP, 2.0% 4 3, Form A 3 35 μL BID/5 days 4 MPP, 2.0%BID = twice a day

Female Gottingen mini-pigs were used in these studies. Animals receiveda single topical ocular dose in the right eye twice daily, approximately12 hours apart (±1 hour), for 4 consecutive days; on the fifth dayanimals received a single topical ocular dose in the a.m. only for atotal of 9 doses over the study duration.

All animals were euthanized with sodium pentobarbital and bloodcollected via cardiac puncture into tubes containing K₂EDTA andcentrifuged to obtain plasma. Then, both eyes were enucleated, flashfrozen and stored at −70° C. for at least 2 hours. Within approximately2 days, the frozen matrices were collected as right and left eye forchoroid and retina.

The resulting drug exposures in plasma and in the back of the eye areshown in FIGS. 8-10 . These results demonstrate that topicalinstillation of crystalline Form A of Compound 3 as MPP results in drugexposure in the retina and choroid in vivo.

EQUIVALENTS AND SCOPE

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

What is claimed is:
 1. A method of inhibiting aberrant vascularendothelial growth factor (VEGF) signaling comprising administering to asubject in need thereof a therapeutically effective amount ofcrystalline form A of7-(3-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinazolin-7-yloxy)propyl)-2-oxa-7-azaspiro[3.5]nonanehaving an X-ray powder diffraction (XRPD) pattern with peaks at about6.11±0.3, 9.63±0.3, 9.87±0.3, 11.10±0.3, 11.46±0.3, 12.26±0.3,12.88±0.3, 14.40±0.3, 15.45±0.3, 15.66±0.3, 16.41±0.3, 17.54±0.3,18.16±0.3, 18.60±0.3, 19.51±0.3, 20.36±0.3, 21.12±0.3, 21.14±0.3,22.31±0.3, 23.01±0.3, 24.79±0.3, 25.71±0.3, 26.84±0.3, and 28.90±0.3degrees two theta.
 2. The method of claim 1, wherein the crystallineform is administered topically, by injection, to the eye, orally, or byinhalation.
 3. The method of claim 1, wherein the inhibition of aberrantVEGF signaling treats an ocular disease.
 4. The method of claim 3,wherein the ocular disease is retinopathy, age-related maculardegeneration, corneal neovascularization, diabetic macular edema, orretinal vein occlusion.
 5. The method of claim 1, wherein the aberrantsignaling of VEGF is associated with cancer.
 6. A method of inhibitingaberrant vascular endothelial growth factor (VEGF) signaling comprisingadministering to a subject in need thereof a therapeutically effectiveamount of crystalline form A of7-(3-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinazolin-7-yloxy)propyl)-2-oxa-7-azaspiro[3.5]nonanehaving an X-ray powder diffraction (XRPD) pattern with peaks at about7.70±0.3, 13.53±0.3, 9.87±0.3, 10.69±0.3, 12.88±0.3, 14.40±0.3,15.45±0.3, 16.42±0.3, 17.27±0.3, 18.44±0.3, 18.90±0.3, 19.73±0.3,21.14±0.3, 22.56±0.3, 23.10±0.3, 26.07±0.3, 26.84±0.3, and 29.12±0.3degrees two theta.
 7. The method of claim 6, wherein the crystallineform is administered topically, by injection, to the eye, orally, or byinhalation.
 8. The method of claim 6, wherein the inhibition of aberrantVEGF signaling treats an ocular disease.
 9. The method of claim 3,wherein the ocular disease is retinopathy, age-related maculardegeneration, corneal neovascularization, diabetic macular edema, orretinal vein occlusion.
 10. The method of claim 6, wherein the aberrantsignaling of VEGF is associated with cancer.